Mechanical circulatory support and heart transplantation are established surgical options for treatment of advanced heart failure. Since the prevalence of advanced heart failure is progressively increasing, there is a clear need to treat more patients with mechanical circulatory support and to increase the number of heart transplantations. This narrative review summarizes recent progress in surgical treatment options of advanced heart failure and proposes an algorithm for treatment of the advanced heart failure patient at >65 years of age. Survival with heart failure (HF) has significantly increased in the last decades with the broad implementation of guidelines-directed medical therapy . NonetheThe prognosis of advanced-stage HF remains poor with a 1-year mortality ranging from 25% to 75% ,7. OftenIn contrast, temporary MCS treatment provides an option to bridge patients with refractory symptoms to long-term mechanical circulatory support (LT-MCS) or heart transplantation (HTx), which remain the two acknowledged options of surgical treatment of advanced HF. While HTx can still be considered as the gold-standard treatment of advanced HF, organ-donor shortages limit the broad application of this treatment option. Whether application of less-stringent criteria for donor selection can increase and expand the donor pool without repercussion on posttransplant survival remains to be followed. On the other hand, LT-MCS therapy has seen a significant surge of activity in the last years, in particular since smaller blood pumps became available . Since tThis review intends to discuss the current evidence for temporary as well as durable MCS treatment and options to expand the donor heart pool for transplantation, and finally, it proposes an algorithm of how to care for the older-age patient presenting with advanced HF.Acknowledging that evidence guiding therapeutic decision making in patients with advanced HF remains scarce , the EurThe case against broad application of traditional inotropes is their potential to increase myocardial ischemia and to provoke tachyarrhythmia, while inotropes improve central and peripheral hemodynamics, reduce congestion, and alleviate end-organ dysfunction on the other hand ,11. TempTemporary MCS can be provided as bridge to recovery (BTR) or bridge to the decision (BTD) if it is not clear whether the clinical situation may offer future eligibility for durable treatment such as long-term MCS (LT-MCS) or HTx. If the patient is not eligible for either option because of ongoing severe infection, uncontrolled bleeding, or larger dekubitus ulcer, temporary MCS treatment can be maintained until the patient is recovering from temporary contraindication (bridge to candidacy = BTC). However, if the patient is eligible for HTx, temporary MCS treatment can be maintained as a bridge to urgent HTx (bridge to transplant = BTT) ,13. AddiMore recent studies provide helpful evidence for decision-making in advanced HF affording temporary MCS treatment and will be therefore discussed in the following.The intra-aortic balloon pump (IABP) is a catheter-mounted balloon sitting in the descending aorta. It augments the pulsatile blood flow by inflation during diastole, thereby displacing blood volume to the distal aorta. Simultaneously, inflation displaces blood to the proximal aorta, increasing thereby the mean pressure in the ascending aorta and the coronary arteries. On the other hand, deflation during systole reduces the afterload . IntuitiHowever, IABP treatment has been applied in recent times more frequently for treatment of advanced HF, especially among patients awaiting HTx. Initially, this application was likely a response to the 2018 United Network for Organ Sharing (UNOS) policy change, which prioritizes patients to status 2 when bridged to heart transplantation with IABP . SurprisThe IMPELLA device received in 2016 Food and Drug Administration approval for use in acute myocardial infarction complicated by cardiogenic shock, but its effect on clinical outcomes has remained controversial . HoweverFor the latter reason, the IMPELLA has been increasingly used as a temporary bridge among decompensated advanced HF patients awaiting heart transplantation . HoweverVA-ECMO remains the most utilized option for temporary MCS in patients with cardiogenic shock. It is a low-cost percutaneous device that allows for rapid implantation and provides full biventricular and pulmonary support. VA-ECMO increases the LV afterload, results in LV dilatation, and augments the incidence of thrombotic events due to stasis of the left-ventricular blood flow, which intuitively suggests an increase of adverse effects with VA-ECMO treatment. However, in the only randomized controlled trial, the ECLS-SHOCK trial, VA-ECMO treatment did not impact negatively on left-ventricular function in 30 days survivors of acute myocardial infarction complicated with cardiogenic shock . In addiVA-ECMO treatment is therefore often applied in combination with left-ventricular (LV) decompression strategies such as IABP and IMPELLA treatment in order prevent LV dilatation resulting from increased LV afterload ,32. In fIn summary, evidence from prospective randomized-controlled trials evaluating temporary MCS with conventional guideline-driven treatment is missing, but trials are ongoing in STEMI patients with concomitant cardiogenic shock  ,37 or caApplication of milrinone or dobutamine in advanced HF failed to show a decrease in morbidity or an increase in survival ,41. HoweIn theory, 10–25% of all advanced HF patients should qualify for LT-MCS treatment taking into account limitations related with age, comorbidity, or social constraint . In realAs with other treatments, the best selection of the suitable candidate is primordial for LT-MCS treatment ,49. TherWhile these specifications identify potential candidates for assist-device implantation, further stratification of advanced HF into seven different levels has proven useful for evaluating the urgency of LT-MCS implantation . Table  This strFor the moment, assist-device treatment in Switzerland is largely reserved for HTx candidates worsening their clinical condition towards INTERMACS level 2–4, similar to other countries. However, the largest growth in LT-MCS treatment in the last years has been seen in advanced HF patients not suitable for HTx. These DT patients present a higher burden of comorbidity when compared to BTT patients, which can explain their higher mortality  as shownWith the growing experience and the progressively improving performance of LT-MCS, former limitations to LT-MCS such as age, BMI, frailty, or renal dysfunction are no longer considered strict contraindications to LT-MCS treatment. In particular, 14% of LT-MCS patients were >70 years old in the MCS Network Research, and their unadjusted survival was 75% and 65% at 1 and 2 years, respectively, while being 84% and 73% in younger patients . HoweverDespite all of this improvement, the right ventricle (RV) remains a point of concern since venous blood return will significantly increase after LT-MCS implantation, whereas unloading of the LV will induce a leftward septal shift. This results in an increase in the end-diastolic RV volume with a subsequent change in its geometry and will ultimately compromise RV function. These changes occur independent of preexisting RV function but are more likely to incidence when RV dysfunction preexists. RV dysfunction can also explain the higher incidence of postoperative bleeding, renal insufficiency, and prolonged length of hospital stay after LT-MCS implantation . PreoperIn the past, more important RV dysfunction was considered a contraindication to LT-MCS. However, post-operative right-ventricular-acute MCS (RV-AMCS) devices have become more commonly used in the clinical setting of pre-operative RV dysfunction. Furthermore, designated algorithms help to select the proper device and provide recommendations for patient monitoring and weaning protocols . In the Finally, aortic-valve regurgitation affords careful evaluation since > mild aortic regurgitation mandates biological valve placement or application of a central leaflet coaptation stitch. However, both approaches increase the perioperative risk of RV dysfunction due to the need of extracorporeal circulation support in the operating room. However, >mild AR affords pre-implantation intervention because an increase in the severity of aortic regurgitation will result in a circulatory short-circuit between the CF-LVAD, the ascending aorta, and the left ventricle. Furthermore, a mechanic aortic prosthesis should be replaced by an aortic bioprosthesis, while a functional aortic bioprosthesis can remain in place .In summary, the arrival of the last generation of continuous-flow LT-MCS together with the experience gained in the last years has expanded the pool of advanced HF patients who will benefit from LT-MCS treatment taking into account the remaining limitations .Heart transplantation (HTx) remains nonetheless the gold-standard treatment for advanced heart failure treatment despite the favorable results of durable MCS with recent continuous-flow LT-MCS . The facThese antidromic developments may result from differences in the waitlist policy. In fact, the U.S. replaced more recently their long-standing three-tier system by a six-tier system based on the deliberation that increased granularity should improve waitlist grading of HF severity, and, thus, better identify the HTx candidate in need of urgent transplantation . The waiCare for congenital heart disease (CHD) has significantly improved in the last decades with more than 90% of children born with these defects surviving until adulthood. HF is the leading cause of death in the majority of these adult patients, and more and more CHD are candidates for HTx  with benp < 0.001) [Despite the clear need for an increased number of donor organs, the utilization rate of donor hearts has remained fairly low worldwide. The reasons for refusal of donor hearts in the U.S. have been a size mismatch, histo-incompatibility, older age, female donor gender, or donor-comorbidities . Guideli< 0.001) . On the < 0.001) . AltogetThe number of HTx candidiates with allosensitization at a high titer of pre-formed HLA-antibodies has doubled over the last decades ,87 and bTM, which maintains a target temperature and avoids placing the donor organ in close proximity to ice, was presented [TM enables longer cold ischemic transport times [Preservation and storage of the donor organ has become an important issue in today’s allocation since traveling distances are larger, prolonging cold ischemic times. In order to decrease myocardial damage from the unmet metabolic need during transport, a cardioplegic solution is applied at procurement to induce diastolic arrest. In addition, hypothermia during transport is maintained until reperfusion in the recipient . Traditiresented . Case rert times , howeverrt times .TM has been evaluated as another option for the transport of the donor organ to the recipient. The monitoring of the donor graft function is possible via visual inspection of the donor heart, measurement of the coronary blood flow, and serial arterial or venous lactate measurements. The PROCEED II trial  randomized 130 patients to standard cold storage or the OCSTM, and the results showed non-inferiority for the latter without a difference in 30 day patient and graft survival [TM without increasing the risk of cardiac-related serious adverse events. The OCS has also been tested for the use of extended criteria donors in the OCS Heart EXPAND trial. Donor hearts in this study were of older donor age, presented left-ventricular dysfunction or hypertrophy, or were donor hearts after prolonged cardiac arrest, exposed to prior drug abuse, or presenting mild to moderate coronary disease. Application of the OCS resulted in an excellent short-term post-transplant survival  and low rates of primary graft dysfunction (10.7% rate of severe left- or right-ventricular dysfunction at 24 hours posttransplant) [TM were successfully transplanted with 5/26 donor hearts discarded because considered unsuitable while on the OCSTM. These 21 patients presented an overall lower burden of postoperative complications when compared to control-group marginal donor hearts preserved by cold storage [TM had favorable short-term results after HTx. However, knowledgeable care of the ex vivo organ is primordial, and this is only provided with routine application of OCSTM-based preservation. In Switzerland, application of the OCSTM faces financial limitations at the moment, especially because of expensive consumables. However, transport times up to 10 hours with successful subsequent HTx have been reported when donor hearts were procured and preserved using this technique [In recent years, continuous, normothermic perfusion with warm, oxygenated, nutrient-enriched donor blood provided by the TransMedics Organ Care System (OCS)survival . Of notensplant) . Another storage . Altogetechnique ,107. TheIn summary, a recent advance in the field of cardiac transplantation provides solutions for the growing demand for suitable organs. Improved size matching considering the heart mass has the potential to improve donor-recipient matching and to reduce the refusal rate based on mismatches of height, body weight, and gender, thereby expanding the donor pool. Furthermore, an advance in organ preservation permits not only procurement at larger distances but also ex vivo appraisal of the function of the donor heart meeting extended criteria for procurement with the potential to increase the number of donor hearts suitable for transplantation. The future of cardiac transplantation will rely on these advances so that a maximum of heart transplant recipients has the chance to derive the best quality of life and survival.Since 2003, the University Hospitals of Geneva and Lausanne have collaborated in a common HTx program with the transplant operation and the immediate postoperative care provided by the latter hospital. In this context, the two centers have agreed on an algorithm paving the treatment of the advanced HF patient >65 years of age. Acknowledging that the indications for surgical care of the younger advanced HF patient are set out clearly by the International Society of Heart and Lung Transplantation and the European Association of Cardiothoracic Surgery ,108, theTM indicate that expansion of the donor heart pool beyond older age and by inclusion of donor heart with structural pathology is possible [The 10 year update of the listing criteria for HTx recommends that advanced HF patients can be considered for HTx if they are <71 years of age, while HTx remains an option in selected patients >70 years . The ovepossible .Nonetheless, the usual option of surgical treatment of an advanced HF patient aged between 65–69 years is DT , especiaThe arrival of the last generation of continuous-flow LT-MCS and the experience gained in the last years has increased the number of advanced HF patients eligible for LT-MCS treatment. This evolution is of importance in particular because of the progressively increasing number of advanced HF patients presenting with older age where HTx does remain an option for highly selected cases. However, the improvement in matching metrics between donors and recipients has the potential to expand the donor pool, resulting in transplantation for otherwise-refused donor hearts. In addition, recent development of organ preservation permits not only procurement at larger distances but also appraisal of extended-criteria donor hearts and, thus, has the potential to increase the number of donor hearts suitable for transplantation. Therefore, there is promise that more patients with advanced HF may have the benefit of surgical treatment of advanced HF already in the near future.


Wiskott–Aldrich syndrome (WAS) is an X-linked genetic disorder with a variable phenotypic expression that includes thrombocytopenia, eczema, and immunodeficiency. Some patients may also exhibit autoimmune manifestations. Patients with WAS are at increased risk of developing malignancies such as lymphoma. Allogeneic hematopoietic cell transplantation remains the only curative treatment. Haploidentical bone marrow transplantation (haplo-BMT) with post-transplant cyclophosphamide (PT-CY) has more recently been applied in WAS. Here, we report two brothers who underwent successful T-cell replete haplo-BMT with PT-CY at ages 9 months and 4 years using their father as the donor. Our myeloablative regimen was well-tolerated with minimal organ toxicity and no acute or chronic graft vs. host disease (GvHD). Haplo-BMT may be considered as a safe and effective option for patients with WAS who do not have available human leukocyte antigen (HLA) matched donors. Wiskott–Aldrich syndrome (WAS) is an X-linked disorder characterized by the triad of microthrombocytopenia, eczema, and immunodeficiency, though all three are not required for diagnosis, as some patients may present with partial or variable phenotypic expression –3. PatieAllogeneic hematopoietic cell transplantation (HCT) remains the only curative treatment for WAS. Matched sibling donor (MSD) transplantation has resulted in event-free-survival > 90%, compared to 70–90% in matched unrelated donor (MUD) and umbilical cord blood (UCB) and 55–90% in mismatched unrelated donor transplantation (MMUD) , 8–11. W9/L. He was admitted for presumed immune thrombocytopenic purpura (ITP) and received intravenous immunoglobulin (IVIG) with a marginal response in his platelet count. Given the persistent eczema and thrombocytopenia with a low mean platelet volume, genetic testing was performed, which confirmed missense mutation, 291G>A, in the WAS gene (R86H). He was referred to our hematopoietic cell therapy and transplant program when he was 17 months of age for bone marrow (BM) transplant evaluation. No HLA-matched related or unrelated donor was identified. As his mother was pregnant, the parents indicated that they wished to wait in the event that the upcoming sibling may be a match. In the interim, the patient received monthly infusions of IVIG and IV pentamidine for Pneumocystis jirovecii prophylaxis while his platelet count ranged between 30 and 40 × 109/L.The older brother (patient #2) presented for his 1-year well child visit with eczema and purpura, and was found to have a platelet count of 5 × 109/L, but he then developed transfusion refractory thrombocytopenia (1–5 × 109/L), which did not respond to IVIG, steroids, or eltrombopag (Patient #1 (younger brother) was born 2 years after his brother. While he proved to be an HLA match, he unfortunately had thrombocytopenia and genetic testing confirmed the same mutation in the WAS gene. In his first 7 months of life, his platelet count ranged between 50 and 80 × 10rombopag . He had 2 IV on days −5 to −2, and melphalan (MEL) 100 mg/m2 IV on day −2  1 mg/kg IV q 6 h for 12 doses on days −8 to −6, fludarabine (FLU) 30 mg/mn day −2 . Busulfan day −2 . BM stem9/L. Antifungal prophylaxis with voriconazole was initiated following completion of BU administration. For P. jirovecii prevention, IV pentamidine was given until 1 year after BMT. Acyclovir was also started on admission for herpes simplex and varicella virus prophylaxis. Bi-weekly polymerase chain reaction (PCR) monitoring for cytomegalovirus (CMV) and weekly for adenovirus, Epstein–Barr virus (EBV), and human herpes virus-6 (HHV-6) were performed until discharge from hospital and subsequently at least every other week during the first 100 days and at least monthly for another 6 months.Standard bacterial prophylaxis was used (Lefofloxacin) when ANC dropped below 0.5 × 109/L was achieved for three consecutive days. Whole blood donor chimerism was evaluated on days +28, +100, +180, and +365. Since both patients had complete donor chimerism, lineage-specific chimerisms were not performed routinely but only at last follow-up, specifically at 27 months for patient #1 and at 12 months for patient #2 (Granulocyte-colony stimulating factor (G-CSF) was started on day +5 at 5 μg/kg/day until an absolute neutrophil count (ANC) of 2.5 × 10tient #2 .Patients and transplant characteristics are summarized in 2 and rabbit ATG had stable engraftment and were alive. The incidence of GvHD was not reported specifically for the WAS patients, but they represented more than a third of a group that had 36% grade II–IV aGvHD and 17% cGvHD. Using BU 16 mg/kg and a slightly lower FLU dose (150 mg/m2) and rabbit ATG, Yue et al. reported five WAS patients from China  than the aforementioned regimens, as MEL is added to the last day of FLU. As indicated above, this regimen was well-tolerated with minimal organ toxicity limited to mucositis, trilineage engraftment with complete donor chimerism, no acute or chronic GvHD, no bacterial infections in the first 3 months post-BMT, and no fungal infections or viral reactivations. BU-based MAC conditioning regimens are more commonly used in WAS with MSD, MUD, MMUD, and UCB transplants and found to be associated with decreased graft failure and lower incidence of mixed chimerism when compared to RIC (We present two brothers who underwent a T-cell replete haplo-BMT with PT-CY from their father. The siblings received MAC, which we have previously reported in haplo-BMT with PT-CY for hematologic malignancies and consists of BU-FLU-MEL but with the addition of rabbit ATG –23. Our d to RIC –10. Thisd to RIC , 18. Whid to RIC –26.As WAS is an X-linked disorder, with an incidence of 1–4 per 1 million males, it would not be uncommon to have more than one boy in a family with this diagnosis. Our experience is unique as there have not been other reports of two brothers with WAS receiving haplo-BMT from the same donor, in this case their father. Moreover, it illustrates that haplo-BMT from the same donor, preferably the father, can be readily performed and be curative. While mothers, who are WAS carriers, have been used in some cases, haploidentical fathers or brothers are preferable. Additionally, the risk of GvHD is believed to be lower when male donors are used for male recipients compared to female donors. It is believed that female donor T cells may react to minor histocompatibility antigens encoded by genes present in the Y chromosome .Our patients were of mixed race, their two sisters were not an HLA match, and unrelated searches failed to identify suitable matches as is the case more often than not for minority populations . ConsideThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Subjects Protection Program The University of Arizona. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.EK wrote the manuscript and managed the patients during their hematopoietic cell transplantation. JS and JA reviewed the clinical records and co-wrote the manuscript. NR and PA edited the manuscript and treated the patients before transplantation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rana sylvatica, is the best-studied of a small group of amphibian species that survive whole body freezing during the winter months. These frogs endure the freezing of 65–70% of their total body water in extracellular ice masses. They have implemented multiple adaptations that manage ice formation, deal with freeze-induced ischemia/reperfusion stress, limit cell volume reduction with the production of small molecule cryoprotectants  and adjust a wide variety of metabolic pathways for prolonged life in a frozen state. All organs, tissues, cells and intracellular organelles are affected by freeze/thaw and its consequences. This article explores mitochondria in the frozen frog with a focus on both the consequences of freezing  and mitigating defenses  in order to identify adaptive strategies that defend and adapt mitochondria in animals that can be frozen for six months or more every year. A particular focus is placed on freeze-responsive genes in wood frogs that are encoded on the mitochondrial genome including ATP6/8, ND4 and 16S RNA. These were strongly up-regulated during whole body freezing (24 h at −2.5 °C) in the liver and brain but showed opposing responses to two component stresses: strong upregulation in response to anoxia but no response to dehydration stress. This indicates that freeze-responsive upregulation of mitochondria-encoded genes is triggered by declining oxygen and likely has an adaptive function in supporting cellular energetics under indeterminate lengths of whole body freezing.The wood frog,  Every species on Earth is endowed with a set of biochemical and physiological strategies that can optimize their survival and reproductive capacity in the face of biotic and abiotic stresses. One of the most amazing survival strategies in the animal kingdom is freeze tolerance, the capacity of numerous invertebrate and a few vertebrate species to withstand ice formation within extracellular fluid spaces of their bodies while resisting intracellular freezing. This is a crucial adaptation for winter survival of many terrestrial ectotherms living in seasonally cold climates ,2. FreezFreeze/thaw is an event comparable to ischemia/reperfusion. Ice formation in extracellular and extra-organ spaces cuts off oxygen delivery to tissues. Hence, cells must use “survival mode” strategies including a switch to fermentable fuels , accumulation of lactate as an end product, endurance of a slow but steady decrease in ATP levels and energy charge, and a necessary suppression of many ATP-expensive cellular activities ,3. As inRana sylvatica.Nowhere else in the cell is the effect of oxygen limitation felt more keenly than in the mitochondria, where the crucial function of the organelle in oxygen-dependent ATP production is suppressed or fully shut down in the frozen state with potentially grave consequences for the multitude of ATP-requiring cellular functions. Furthermore, freeze-induced disruption of mitochondrial functions can potentially trigger apoptosis. The present article explores the role of antioxidants and the adaptive responses of mitochondria to whole body freezing, summarizing research carried out on the primary vertebrate model of freeze tolerance, the wood frog, Terrepene carolina) and European common lizards (Lacerta vivipara) [Among vertebrates, freeze tolerance is most common among amphibians that live at high latitudes or altitudes. Several species of frogs and two Siberian salamander species are known to be freeze tolerant and capable of surviving days, weeks or months of continuous freezing . Hatchliivipara) .Rana sylvatica (recently renamed Lithobates sylvaticus), is the best-studied of the freeze tolerant vertebrates and the main model used to explore molecular, biochemical and physiological mechanisms of freezing survival [The wood frog, survival ,2. This survival . Speciessurvival ,2. R. sylvatica tissues as compared with a comparable species that winters under water in the same locale, the leopard frog Rana pipiens. The same was true of superoxide dismutase, glutathione S-transferase and glutathione reductase; enzyme activities in wood frog tissues were generally about twice as high as in leopard frogs [R. pipiens tissues [Studies of freeze tolerant animals can reveal information about how mitochondria respond to the very long interruptions of oxygen supply and the readjustments needed to deal with days, weeks or months of life in a frozen state. One biochemical adaptation that we have identified is the maintenance and/or upregulation of antioxidant defenses . For exard frogs . Further tissues ,7. These tissues .R. sylvatica [Another study assessed changes in protein levels of five antioxidant enzymes (using a 5-plex Luminex Oxidative Stress Magnetic Bead panel) to compare responses to three stresses  in the brain and heart of ylvatica . Note thylvatica . CatalasSimilar context-specific patterns were found using a 5-plex Heat Shock Protein panel. Of particular interest, was the upregulation of Hsp60  in anoxic brain by 1.7-fold in response to 4 or 24 h of anoxia and by 2.1-fold in the heart of 4 h frozen frogs but with very little change in cytosolic HSPs . ComparaThe responses of NF-κB, an oxygen-sensitive transcription factor, to wood frog freezing also support the proposal that enhanced antioxidant defenses contribute to cell protection in the frozen state. Under normal unstressed conditions, NF-κB is held in the cytoplasm by its inhibitory protein, IκB, but when ROS levels rise, IκB is phosphorylated, ubiquitinated and targeted for degradation, leaving NF-κB free to translocate into the nucleus and upregulate antioxidant genes . ProteinTrachemys scripta elegans) with upregulation of both transcript and protein levels of the p50 and p65 subunits. The response was particularly strong after 5 h of anoxic submergence but returned to near baseline after 20 h submergence [Upregulation of antioxidant defenses is also crucial for endurance of environmental stress in turtles to deal with anoxia/hypoxia stress in adult turtles during diving or underwater hibernation  or freezmergence . DNA-binmergence . MnSOD sChrysemys picta marginata). Both transcript and protein levels of Nrf2 were elevated in several tissues after 5 h of freezing of hatchlings and six isozymes of glutathione S-transferase  increased significantly (by 1.5–2.5 fold) in selected tissues, primarily in the brain [The action of another transcription factor involved in antioxidant defense, Nrf2, responded to whole body freezing in hatchling painted turtles  and lower Km H2O2 (0.64 fold) compared with catalase from control (5 °C acclimated) frogs [m value. Properties of both cytoplasmic Cu/Zn-dependent superoxide dismutase (CuZn-SOD) and mitochondrial Mn-dependent SOD (MnSOD) also changed in wood frog skeletal muscle during freezing [max in muscle from frozen frogs but no significant change in kinetic parameters whereas MnSOD from muscle of frozen frogs showed increased affinity for substrate and reduced sensitivity to urea denaturation, as compared with controls. Total phosphorylation of MnSOD also increased ~2.8-fold for the muscle enzyme from 24 h frozen frogs, comprised of 2.4- and 1.3-fold increases in serine and tyrosine phosphorylation of the protein. This indicates that substantial posttranslational modifications can stabilize/adapt MnSOD to deal with an altered intramitochondrial environment in the frozen state. Not only are the activities of various antioxidant enzymes increased in wood frog tissues in response to freezing but enzymatic properties can also be modified by posttranslational modifications. For example, skeletal muscle catalase from frozen (−2.5 °C) wood frogs showed a significantly higher Vd) frogs . These cfreezing . Cu/Zn S2O2 breakdown) have damaging effects on cell proteins and membrane phospholipids. However, H2O2 also acts as a signaling molecule that is exported from mitochondria to trigger and coordinate responses by a variety of extra-mitochondrial cell functions, both protective and destructive [Both superoxide radicals and hydroxyl radicals , that supplies the Pi needed for ATP synthesis, were strongly upregulated by freezing in wood frog liver [Several freeze-upregulated genes code for proteins that function within mitochondria or are genes that are coded on the mitochondrial genome. An example of the former is the adenine nucleotide translocator  that is a highly abundant protein located in the inner mitochondrial membrane. Screening of a cDNA library made from liver of freeze-exposed (−2.5 °C for 24 h) versus control (5 °C-acclimated) wood frogs confirmed ANT as freeze-upregulated [og liver . Overallog liver . HSP60 iC. picata marginata. Upregulation of several proteins associated with antioxidant defense (by 1.6- to 3.5-fold) was identified for iron binding proteins  and antioxidant enzymes  [Changes in the proteome of wood frog liver between summer and winter have also been identified using liquid chromatography coupled with tandem mass spectrometry quantitative isobaric peptide mapping . Proteinredoxin) , furtherThe above sections dealt mainly with measures that can protect or stabilize mitochondria during freezing in wood frogs. However, adaptive regulation of the activities of mitochondrial enzymes also deserve consideration. Although oxygen depletion will ultimately halt or strongly restrict oxygen-dependent mitochondrial functions , regulatory controls on selected enzymes could still mediate or mitigate other metabolic functions of mitochondria.For example, various aspects of amino acid metabolism continue in the frozen frog. Cellular energetics are supported by anaerobic metabolism in frozen frogs with the accumulation of both L-lactate and L-alanine as glycolytic end-products ,30. AlanWood frogs also build up urea in tissues during freezing and this small molecule supplements glucose as a colligative cryoprotectant. Urea is a known osmoprotectant in amphibians that undergo dehydration stress. In particular, numerous frogs and toad species enter a hypometabolic state called estivation in the hot dry summer months. During this time they undergo progressive muscle atrophy as they catabolize muscle protein for fuel while utilizing the amino groups released to produce urea that is retained as a colligative deterrent to water loss . Hence, + → α-KG + NH4+ + NADH + H+), the liver enzyme from frozen frogs, compared with controls, showed a 39% higher activity of GDH. Km values for glutamate and NAD+ were both ~60% lower (indicating greater affinity for these substrates) and inhibition by GTP was greatly reduced [4+ production to feed urea synthesis. GDH from liver of control versus frozen frogs also differed in posttranslational modifications with the enzyme from liver of frozen frogs showing reduced acetylation and ADP-ribosylation but elevated lysine methylation as compared with controls [A key player in amino acid catabolism and urea synthesis is glutamate dehydrogenase (GDH), its reversible reaction allowing GDH to act as both a nitrogen donor and acceptor. Catabolism of several amino acids  ends via GDH with the product, alpha-ketoglutarate (α-KG), funneled into the Krebs cycle and the ammonium ion either excreted or used for urea synthesis. An analysis of purified wood frog liver GDH showed multiple properties that could promote amino catabolism in support of urea synthesis. For example, when assayed at 22 °C in the direction of glutamate catabolism . CP is then attached to ornithine via OTC to form citrulline and exported to the cytoplasm where the remaining reactions of cycle are completed, ending in urea output. Purified CPS1 from liver of frozen versus control frogs showed significant changes in properties including a higher maximal activity, greater affinity for NH4+, reduced sensitivity to urea inhibition and, in the presence of high glucose, greater affinity for ATP and the obligate CPS1 activator, N-acetylglutamate. CPS1 from control and frozen frogs also differed in posttranslational modification by lysine glutarylation that was significantly reduced on the enzyme from frozen frogs [Crucial first steps of the urea cycle also take place in the mitochondria catalyzed by carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC). Differential regulation of CPS1, the rate-limiting enzyme of urea synthesis, was detected in the liver of control (5 °C) versus frozen (−2.5 °C) wood frogs . This enen frogs . These rATP6/8, CytB, COX1-3, ND1-6 and ND-4L), 22 tRNAs, 2 rRNA genes and a noncoding control region [Lithobates catesbeianus (that winters underwater), and three ranid species found in northeastern Asia. The mitochondrial genome of another freeze tolerant frog, the North American treefrog Hyla versicolor (recently renamed Dryophytes versicolor) has also been sequenced and The mitochondrial genome is a small closed circular molecule  and contains 13 protein-coding genes (l region . It is wl region . No unus32P-labeled cDNA probes and poly(A)+ mRNA from brain of control frogs (5 °C acclimated) vs. frozen frogs  revealed enhanced expression of ATP synthase 6/8 and 16S rRNA genes during freezing [ND4) [A search for mitochondria-encoded genes that were freeze-responsive in wood frog tissues revealed up-regulation of several genes. Screening of a wood frog brain cDNA library via Northern blotting using freezing . In adding [ND4) .2 and upregulation of P0, enhancing complex formation, can exert inhibitory control on ribosome assembly under low oxygen conditions [Several nuclear-encoded genes were also identified as up-regulated from these same screens including ribosomal protein L7, acidic ribosomal phosphoprotein P0, phosphoglycerate kinase 1 (PGK1), and a novel protein that was named Li16 ,42,43,44nditions . Hence, nditions . PGK1 upnditions . Studiesnditions , a potenR. sylvatica were also identified with interesting results. The mitogenome encodes two subunits (ATP synthase 6 and 8) of the mitochondrial Fo complex  that is part of the FoF1 ATP synthase, also known as complex V of the electron transport chain. These two genes share an unusual characteristic, a 46 nucleotide overlap of their sequences, with ATP8 being coded via the +1 reading frame whereas ATP6 gene is read on the +3 reading frame. Hence, the genes are typically referred to as ATP 6/8. The ATP 6/8 transcript retrieved from a wood frog brain cDNA library  [ATP 6/8 transcript levels were unaffected in skin, heart and skeletal muscle.Freeze-upregulated mitochondria-encoded genes of 1.05 kb)  showed ~ATP 6/8 expression was also independently assessed in tissues from wood frogs exposed to two component stresses of freezing: anoxia (frogs given 24 h under a nitrogen gas atmosphere at 5 °C) and dehydration . Both of these individual stresses are fully survivable by wood frogs [ATP 6/8 responded robustly to anoxia rising within 1 h by ~2.5-fold in the brain  was also upregulated by freezing as determined by screening a liver cDNA library. ND4 protein is one of the seven subunits of NADH-ubiquinone oxidoreductase (complex 1 of the electron transport chain) that are encoded on the mitochondrial genome, all of which are hydrophobic subunits found in the transmembrane portion of the large 44 subunit ND protein. A single band was detected on a RNA blot at ~1.4 kb when loaded with liver total RNA samples and probed with liver clone Li39s insert [ND4 transcripts also increased strongly when frogs were under anoxia exposure but decreased in frogs dehydrated to 40% of total body water lost. Once again, this gives evidence that freeze-induced upregulation of a gene on the mitochondrial genome is a response to oxygen restriction.The mitochondria-encoded gene ND2, ND4 and ATP6 genes in high altitude lineages (>3000 m) of galliform birds as compared with related lowland species. Similarly, Jin et al. [ND2, ND3, ND4, ND5 and ND6 among different Chinese toad-headed lizard species of the genus Phrynocephalus that range over a 4200 m altitudinal gradient. Both of these studies support the idea that small changes in the sequences of mitochondria-encoded proteins (and their expression) could have a crucial impact on hypoxia tolerance and cold hardiness among various amphibian and reptile species living in seasonally cold environments, including those that use strategies of freeze tolerance. This is certainly an idea worth further exploration.The mitochondrial genome is widely used by researchers investigating phylogenetic relationships among species but equally there is interest in determining if changes in the mitogenome aid animals in adapting to environmental pressures that could, in some cases, ultimately define a new species. For example, for birds living at or flying over high altitude locations, hypoxia is unavoidable and yet high rates of aerobic ATP production are needed to power flight. As such, every step in aerobic respiration could undergo selection to increase efficiency as illustrated for cytochrome c oxidase by Scott et al.  in studin et al.  reported16S RNA gene was identified as upregulated in selected tissues of freeze-exposed wood frogs as determined by Northern blotting [The mitochondrial genome also encodes 16S and 12S ribosomal RNA genes  that, toblotting . A stronHyla versicolor (recently renamed Dryophytes versicolor) and the closely related Cope’s gray treefrog (D. chrysoscelis) (diploid) [D. chrysoscelis has been used for extensive studies of glycerol import/export from cells and analysis of the aquaglyceroporin (a modified aquaporin) that conducts this transport [Considerable research has also been done on the biochemical adaptations to freezing used by North American gray tree frogs, both the tetraploid diploid) . Both usransport .H. versicolor  on their expression. Anoxia exposure led to a 2.4-fold increase in cytochrome c oxidase, subunit 2 (COX2) transcript levels in H. versicolor skeletal muscle whereas ND3 transcripts were reduced by 50%. Freezing had somewhat different effects; COX1 transcript levels were significantly reduced to ~60% of the control value and COX2, ND3 and ATP6 transcript levels also decreased by about 25–30%. The common factor in these stress responses was a reduction in ND3 transcripts under both conditions, which might suggest a possible regulatory role for this subunit of the first respiratory chain complex that could help to modulate respiratory chain activity in response to oxygen availability.A study of the mitochondrial genome of rsicolor  has alsoFreeze tolerance is an amazing adaptation exhibited by only a few vertebrate species. Pre-existing well-developed tolerances of anoxia and/or dehydration are well known components of freeze tolerance. The present paper highlighted the major importance of antioxidant defenses to freezing survival and emphasized a new concept, the key role for mitochondria in freezing survival. This is supported by the upregulation of a variety of genes  that code for proteins residing in mitochondria. These proteins can provide either protective actions or support continuing low level mitochondrial ATP production for as long as oxygen reserves permit.


A proinflammatory response has been suggested to be involved in the pathophysiology of depression in a subgroup of patients. However, comprehensive largescale studies on neuroimmunological investigations of the cerebrospinal fluid (CSF) are lacking and no largescale longitudinal CSF studies comparing patients with depression to healthy controls currently exist.Primary outcomes: CSF white cell count, CSF/serum albumin ratio, CSF total protein levels, IgG index, CSF levels of IL-6 and IL-8, and the prevalence of any CNS-reactive autoantibody in CSF and/or blood. Secondary outcomes: exploratory analyses of a wide range of neuroimmunological markers and specific autoantibodies. Power calculations are computed for all primary outcomes based on previous CSF studies including patients with depression and healthy controls.A longitudinal case-control study including at least 100 patients with first time depression (ICD-10: F32) within the past year with ongoing symptoms and at least 100 sex and age matched healthy controls with collection of CSF, blood, and fecal samples. All individuals will be evaluated by neurological examination including neurological soft signs, interviewed for psychopathology assessment and have symptomatology evaluated by relevant rating scales. Level of functioning and quality of life will be evaluated by a panel of interview questions and rating scales, and cognitive function assessed by a relevant test battery. In addition, a large number of potential confounders will be registered . This study will represent the hitherto largest investigation of CSF in patients with recent onset depression compared to healthy controls. We expect to elucidate neuroimmunological alterations in individuals with depression and characterize an immunological profile paving the way for the development of effective treatments based on biomarkers.The study is approved by The Regional Committee on Health Research Ethics  and The Danish Data Protection Agency .The online version contains supplementary material available at 10.1186/s12888-021-03633-0. The underlying pathophysiology of depression is heterogeneous and yet not fully understood. In this study we hypothesize that the pathophysiology in a subgroup of patients is caused by altered immunological activation and/or function, as it has been suggested by several authors –3. This However, the blood is separated from the brain by the blood-brain-barrier and the blood-cerebrospinal fluid (CSF)-barrier - that we hereafter refer to commonly as the blood-brain-barrier (BBB) as this expression is more commonly used in the literature. Measuring of immunological markers in the CSF, the material closest to the brain obtainable from living patients, is the gold standard when conducting studies of neuroinflammation. White cell count (WCC) in CSF is one of the most direct measures of increased immune cell activity within the brain, but is an unspecific indicator of pathological changes, since it can be caused by a variety of disorders . Cytokinn = 104 [n = 90 [n = 30 [n = 29 [Overall previous CSF biomarker studies of patients with depression compared to healthy controls vary in sample size with markers like IL-8 and CSF total protein measured in large cohorts of patients : F32) and a minimum of 100 age and sex matched healthy controls.The aim of this study is to identify neuroinflammatory alterations that contribute to the development of depression by longitudinal examinations of CSF, blood and gut microbiota together with thorough psychopathological and neurological examination leading to a more detailed understanding of the interplay between the immune system and the brain.The publication plan with elaboration on primary outcomes is given in Table To exploratively search for differences between patients and healthy controls in a broad variety of neuroimmunological markers, CSF cytokines and chemokines, and specific analyses of CNS-reactive autoantibodies in CSF and/or blood. The secondary outcomes are specified in Table The study intervention will be conducted at the facility of Biological and Precision Psychiatry, Copenhagen Research Centre for Mental Health (CORE), Mental Health Centre Copenhagen, Copenhagen, Denmark or where the patient is hospitalized. The study intervention will always take place in an undisturbed room. Patients will be offered transport if needed.Patients with a first-time diagnosis of a depressive disorder (according to ICD-10: F32) diagnosed within the past year.Ongoing depressive symptoms..Age between 18 and 50 yearsObtainment of written informed consent.Healthy individual.Age between 18 and 50 years.Obtainment of written informed consent.The healthy controls will preferably be matched with patients in accordance to sex and age.The exclusion criteria are described and explained in Table Patients are recruited from the in- and outpatient facilities at The Mental Health Services of the Capital Region of Denmark, and a majority of the patients will be recruited from the Referral- and Diagnostic Department (RDD). For a more detailed description see eSupplementary paragraph Healthy controls will mainly be recruited via a Danish web portal used to find participants for clinical studies (www.forsøgsperson.dk) and will thus represent community controls originating from the same geographic area as the patients in accordance to the Newcastle-Ottawa quality assessment scale for case control studies (NOS) .All participants are screened according to the eligibility criteria prior to the date of enrolment. They are all informed both orally and in writing prior to the enrolment with sufficient time to consider participation. For a detailed description of this procedure see eSupplementary paragraph For each participant, we aim to perform all clinical measurements and biological samples on the same day. The inclusion starts approximately at 9:00 a.m., but due to feasibility the starting time can fluctuate between 8:00-10:00 a.m. The study program is scheduled to last 3-5 h and will vary depending on symptom severity and the participant’s need for breaks. For a detailed description of the study time schedule see eSupplementary Paragraph Before any examination is carried out, informed written consent for participation is obtained  , includiSome questions have been added to the interview of the participants in order for the interviewers to afterwards be able to rate the participant on the following psychopathology rating scales; 17-item Hamilton Depression Rating Scale (HAMD-17) , 6-item During the visit, all participants will be asked to complete a set of questionnaires including Major Depression Inventory (MDI) , and quaCognitive testing will be done using the Brief Assessment of Cognition in Schizophrenia (BACS) , MontreaThe following self-reported data is collected; height, weight, smoking status, alcohol intake, use of recreational drugs, allergies, somatic illnesses, prior and concurrent psychiatric disorders, current medication (both psychotropic and non-psychotropic), intake of non-steroid anti-inflammatory drugs, paracetamol and antihistamines in the prior 2 weeks, use of antibiotics in the prior six months, information on participants menstrual cycle, time for last intake of food and years of education. Information on prior and concurrent psychiatric disorders and medication status/duration is confirmed by the medical record as well. Information on current diet and exercise habits will be provided from a modified version of a questionnaire  used forTo minimize interrater variability, we will regularly conduct co-evaluation of patients with main focus on rating scales. Two research assistants will participate in a full participant inclusion and will individually be diagnosing and rating the participant. Any discrepancies will be noted and discussed with a specialist in psychiatry.In accordance with the NOS, the gold standard for an adequate case definition is independent validation. All patients will be evaluated by SCAN interview. Additionally, patients who require hospital-based treatment (in- or outpatients) are evaluated by the treating psychiatrist in the clinic before referral to the study. Patients who are not treated at in- or outpatient facilities of the psychiatric hospitals (e.g. followed by a private psychiatrist) will be either independently evaluated by an experienced psychiatrist who will watch the video recording of the SCAN interview or by consensus diagnosis with an experienced specialist in psychiatry. The SCAN interviewer will not be blinded to patient status or diagnosis.The patients will be invited to a follow-up visit after one to two years after enrolment. This follow-up visit is planned to include all the same measurements (including blood and CSF measurements) as for the primary visit and a Questionnaire about the Process of Recovery (QPR) will be added . By the To minimize the impact of diurnal variation all samples are to be collected between 8.30 a.m. and 01.00 p.m.Venous blood samples will be collected prior to lumbar puncture and we aim to have ≥90% of blood samples collected between 9.30 and 11.30 a.m. Prior to the blood sample, a skin swab is performed and stored at −80°C for quality control analyses. A maximum of 44 mL of blood will be sampled; 8 mL used for initial analyses and 36 mL stored for later use. For a detailed description of blood sample drawing and equipment, see eSupplementary Paragraph 4.The lumbar puncture will be carried out according to current consensus , and we Thirty-six mL of blood and 14 mL of CSF will be used for biobank storage and will be stored both on filter papers and portioned in 0.5 ul aliquots after centrifuging at 1145 G for 10 min. For further details of blood and CSF storage procedures, see eSupplementary Paragraph In order to assess the risk of adverse events associated with lumbar puncture, participants are contacted on the day after the intervention. All symptoms that could be due to the procedure are noted with specific focus on possible post lumbar headache and infections.Each participant is asked to provide a fecal sample, either during the inclusion, or afterwards from home. Fecal samples are collected in two ways, both in a Sarstedt tube  and by using the OMNIgene•GUT OMR-200 Sample Collection Kit . If collected at home, the fecal samples will be sent by mail, and then stored in the freezer at − 80 degrees Celsius. Samples done during the visit will be frozen immediately. A score on the Bristol Stool Scale , time anBlood pressure and pulse will be measured prior to the neurological examination.We will use the Danish registers to obtain information on socioeconomic factors and prior, current and future medical history, such as prior infections treated in the primary care sector, comorbidities, treatment outcome  and later psychiatric diagnoses.All data will be kept in secured folders and in the database RedCap , 64 in aAll cryo vials will be placed at − 80 degrees Celsius as soon as possible, and time for freezing will be noted. The filter papers will be left to dry overnight at room temperature and placed at − 80 degrees Celsius on the next day. For details see eSupplementary Paragraph All analyses will be run blinded by pseudonymization. Laboratory analyses will be performed according to a statistically designed experimental schedule based on a randomized block design or similar, to minimize the risk of confounding by time of analysis, batch and other factors.Eight mL of blood and two mL of CSF will be analyzed immediately at Rigshospitalet. Initial blood analyses include; white blood cell (WBC) count, erythrocytes, hemoglobin, platelets , high sensitivity CRP, IgG , albumin, glucose  and HbA1c . Initial CSF analyses include; WCC , differential count , albumin, IgG, total protein , erythrocytes , glucose and lactate .CSF total WCC, CSF/serum albumin ratio, CSF total protein and IgG index will be analyzed immediately as described above. IgG index is calculated by the following formula:CSF Cytokines are planned to be analyzed using the V-plex Neuroinflammation Panel 1 Human Kit from Meso Scale Diagnostics. CSF and blood CNS-reactive autoantibodies are planned to be analyzed by Autoimmune Encephalitis Mosaic 1 and GAD65 panels from Euroimmun.Primary and secondary outcomes for gut microbiota analysis are yet to be defined due to the fast development of gut microbiota research. However, a detailed analysis plan including well defined primary and secondary outcomes for gut microbiota samples will be conducted prior to finishing the longitudinal collection of samples.t-tests using standardized mean differences (SMDs) and based on not yet published results from a meta-analysis from our group comparing patients with unipolar depression to healthy controls. If all values are observed above the lower limit of quantification (LLOQ), we expect the power for CSF/serum albumin ratio and CSF total protein to be high (95% or higher), whereas the power for IgG index and CSF total WCC are expected to be low (< 50%) though effect sizes in previous studies are heterogenic. For IL-6 we expect good power (> 80%), but low power for IL-8 (< 50%).Power and sample size calculations are based on two-sample Based on previous studies we expect a proportion of biomarker measurements to be lower than LLOQ, most pronounced in the control group. By simulation, we found that given an effect size leading to a power of 80% without censoring, 33% censoring will only reduce power with a maximum of 5%, while 50% censoring will only reduce power with a maximum of 10%, when using the censored Gaussian model . If valp-values. Two-sided tests with p < 0.05 will be considered significant. All analyses will be done in the program R version 4.0.5 [Pearson chi-square test without adjustment for continuity will be applied for analyses of CNS-reactive antibodies. All results of primary and secondary analyses will be reported with the effect size, 95% confidence intervals and on 4.0.5  or lateron 4.0.5  to fit tThe most recent meta-analyses in this field indicate that large-scale CSF studies of depression are needed , 16. As The secondary outcomes will consist of broader and more exploratory analyses of neuroimmunological markers  and autoantibody profiles hereby providing information for a more thorough biological understanding of the etiology/pathophysiology of depression.We intend to study patients with a first-time diagnosis of depression within the past year, since we expect immunological differences to be more pronounced in the early stages and at acute onset of depression. Additionally, this approach enhances our possibilities of evaluating the CSF neuroimmunological biomarkers as prognostic biomarkers. Furthermore, the participants’ trajectories will be assessed by clinical follow-up and follow-up in the nationwide Danish registers.All patients will be interviewed following the SCAN manual and in accordance to NOS we will strive for an independent diagnostic evaluation of > 95% of the patients. SCAN is a well validated and widely used instrument in assessing, measuring and classifying the psychopathology and behavior associated with the major psychiatric syndromes in adult life .We evaluate the severity of depression by interviewer-rated scales (HAMD-17/6 and MADRS-10/6) and by the self-rating scale MDI. A detailed description of symptom rating scales is provided in eSupplementary paragraph We expect some eligible patients to decline participation solely due to the lumbar puncture. This is partly overcome by increasing knowledge of the procedure among patients and hospital staff, since lack of knowledge seems to contribute the most to unwillingness towards lumbar puncture .The intervention has a time frame of approximately 3-5 h introducing the risk of the most severely depressed patients rejecting participation due to the duration of the intervention. This is overcome by breaking the inclusion down in smaller parts performed in different days and offering breaks whenever needed, and it is accepted that the most severely ill patients do not fulfill questionnaires or cognitive testing.Furthermore, it is essential to exclude patients treated with electroshock therapy (ECT), because ECT leads to a substantial proinflammatory response and there is some evidence of induced short-term neuroinflammation . PatientSeveral measures in this study are self-reported, introducing a risk of inaccuracy in data ; however, we do not expect this to differ between the groups. Additionally, we expect most of the self-reported data to be more reliable  and more extensive  than the available register data.CSF is the obtainable material closest to the brain and the gold standard when conducting studies investigating immunological biomarkers of depression, and the collection of CSF samples is a substantial strength of this study. Previous studies of e.g. CSF IL-6 in depression have included far smaller sample sizes compared to this present study and the large sample size of this study is an important strength. Additionally, the thorough psychopathological evaluation and case definition by SCAN and broad symptom evaluation . Furthermore, we expect the most severely ill patients to be less likely to participate (discussed previously). By including only patients with a recent onset depression and, as close to symptom debut as possible, it is aimed to minimize the proportion of included patients treated with antidepressant medication at the time of assessment. However, it is a limitation of this study that some participants are expected to be treated by antidepressants, since antidepressants have been suggested to yield immunomodulatory effects .Lumbar puncture is a commonly used procedure for diagnostics of CNS disorders and serious complications are very rare with the most common side effects being backache, nerve root irritation and headache, while infections are described only in case reports . By usinThe study will be conducted in accordance with the principles of the Declaration of Helsinki , and other applicable laws and regulations. Participants will always be informed of the risks listed under "Risks and side effects" in accordance to the paragraph "Information and screening assessment". It is emphasized that the participant has the right to retract the consent at any time and, for patients, that this will have no influence on future treatment in the psychiatric department/clinic. All individuals will be pseudoanonymized when analyzing the data. The identity of participants will only be used for merging of the data and retrieving biological samples. Data will only be published in a manner where no individuals can be identified.Lumbar puncture is routinely used for diagnosing a variety of disorders, e.g. multiple sclerosis and dementia, associated with depression , 73, andThe project described in this protocol paper is part of the larger PSYCH-FLAME project investigating the immune system’s involvement in psychotic  and affeLarge, well-conducted CSF studies are lacking and will have the potential to improve diagnostics and treatment of individuals currently diagnosed with depression, and to identify new biomarkers associated with the disorders. This study represents the largest, most comprehensive study to date investigating immunological and neurological biomarkers of patients with recent onset first time depression compared to healthy controls, and it will be the first study to investigate these biomarkers longitudinally with the potential to gain new insight into the correlations between symptoms of depression and neuroinflammation.We expect the currently defined group of patients with depression to be divided into subgroups that are more homogenous than the current broad classification of depression. Stratification based initially on CSF biomarkers will subsequently be sought extended to blood-based biomarkers for easier clinical management. This could contribute to a paradigmatic change and pave the way for development of more effective treatments based on biomarkers in some cases, as opposed to only being based on clinical observations.Additional file 1.


Cerebrospinal fluid total protein (CSF-TP) levels in adults with posthemorrhagic hydrocephalus (PHH) are poorly studied. The objective of this study was to explore the characteristics of CSF-TP levels in patients with PHH.The clinical data of 156 patients with hemorrhagic brain disease were retrospectively studied and divided into PHH and NPHH groups. Single-factor and multi-factor analyses were performed, and the key role of CSF-TP was evaluated using linear analysis.p = 0.017), days [total fever time when body temperature ≥ 38.5°C (p = 0.04)], Glasgow Coma Scale (GCS) score (p < 0.001), and time (from the onset of the disease to the obtainment of CSF-TP after lumbar puncture (p < 0.001) were important factors for PHH. Logistic regression analysis revealed that GCS score < 8  and CSF-TP × time ≥ 9,600  were independent risk factors for PHH. All CSF-TP values were averaged every 2 days. CSF-TP was negatively correlated with time. Linear analysis showed that CSF-TP in the PHH group was higher than that in the NPHH group at the same onset time, and that the duration of detectionin the CSF was longer.Among the 156 patients, 85 (54.5%) had PHH and 34 (21.8%) underwent surgeries. Hypertension  × time ≥ 9,600 and GCS score <8 were independent risk factors for PHH. CSF-TP was higher in the PHH group than in the NPHH group. The term posthemorrhagic hydrocephalus (PHH) was first proposed by Murtagh and Lehman in 1967. It initially referred to the progressive dilatation of the brain ventricular system in neonatal intraventricular hemorrhage (IVH) , 3. PHH The objective of this study was to analyze the risk factors for hemorrhagic brain diseases complicated by PHH and explore the relationship between CSF-TP and PHH. This study was approved by the Ethics Committee of Jiangxi Provincial People's Hospital (No.2021-074) and was carried out in strict accordance with the Helsinki Declaration (2013). Relevant medical records were collected retrospectively between January 2013 and October 2020. Univariate and multifactorial analyses of clinical factors were performed to determine the independent risk factor. The relationship between CSF-TP and PHH was evaluated using linear analysis.TBI, SAH, IVH, ICH, etc., clearly diagnosed by CT.Biochemical data of the cerebrospinal fluid were collected during hospitalization.Hospitalization courses or traceable courses are longer than 2 weeks .Hematological diseases, brain tumors, and hydrocephalus were diagnosed before onset.Intracranial infection was diagnosed during the course of the disease.Decompression of bone flap was performed.Concomitant complicated liver, kidney, gastrointestinal, respiratory, cardiovascular, and other diseases that seriously affect safety assessment.Concomitant serious infectious disease.Complications of coagulation dysfunction or long-term use of antiplatelet or anticoagulant drugs.Pregnancy or lactation.All the patients received standardized nursing care and treatment in the neurosurgery department. Our department had a well-trained professional team who strictly followed the guidelines for neurosurgery treatment formulated by China –11. AfteBasic information  was obtained from the inpatient information management system of our hospital, from which past information , detailed treatment information , and laboratory test data  were collected. The diagnosis of PHH was based on the first discharge diagnosis, review results, and the second admission diagnosis. Information collection and statistical analysis were conducted by professional personnel.t-test or Mann–Whitney U-test and chi-square test, and multivariate analysis performed binary logistic regression . For all the tests, p < 0.05 was considered statistically significant.The SPSS software  was used for the statistical analysis, and the statistical tests were bilateral. Regarding the distribution of CSF-TP, the X-axis referred to the time at which CSF-TP was obtained, where the time of disease onset was set at 0. The Y-axis indicated the CSF-TP levels. Statistical classification variables are expressed as counts and percentages (%), and continuous variables are expressed as mean (x) ± SD. Parametric or nonparametric tests were performed according to whether the data conformed to a normal distribution. Univariate analysis was carried out using the Between January 2013 and December 2020, 3,947 hospitalized patients were screened using a case information system for retrospective analysis. Finally, 156 patients were included in the study based on the inclusion and exclusion criteria. The number of patients in each phase is shown . Of the Data on sex, age, diabetes history, days , hypertension history, CSF-TP , and time (from the onset of the disease to the time the CSF-TP was obtained after lumbar puncture) were collected and included in the study .p < 0.05) in the single factor analysis were analyzed by multiple factors   with statistical significance ( = 0.09) .Table 4). CSF-TP × time was statistically significant in the univariate analysis. Therefore, CSF-TP × time was used as a new reference indicator. Then, 1,200 × 8 = 9,600 was taken as the boundary in the multivariate analysis after grouping. The factors were divided into two groups. In binary regression analysis, GCS < 8  and CSF-TP × time   were independent predictors  , as well as the CSF-TP values , the number and proportion of complicated PHH cases were calculated .p = 0.005), hypertension history (p = 0.017), days (p = 0.04), GCS score (p < 0.001), and time (p < 0.001) were unbalanced variables in the entire cohort. Because the value of CSF-TP changes with time, we analyzed the CSF-TP and time variables as a product of each other, and the results showed that the CSF-TP × time index was a significant factor (p = 0.001). After grouping, the binary logistic regression analysis showed that a GCS score <8  and CSF-TP × time   were independent risk factors for PHH. CSF-TP changed gradually with time, and the average TP was obtained by grouping the values every 2 days for statistical linear analysis. The distribution of TP values in the NPHH group was significantly lower than that in the PHH group, with some TP values lower than the normal level, which indicated that the PHH of patients had occurred at this time. The CSF-TP values were divided into two groups . The statistics of the rate of PHH (PHH-R) in the two groups showed that PHH-R increased significantly, and that the >1,200 mg/L group was more obvious  is a common complication of intracranial hemorrhage that can be secondary to ICH, IVH, TBI, or SAH . The morAfter IVH, red blood cells in the bloody CSF rarely increase again but gradually lyse. There have been many studies on lysed products; however, there are few reports on the influence of CSF-TP on PHH. CSF-TP is age-dependent, and its concentration in men is slightly higher than in women at 40 mg/L . A studyCSF amyloid protein, nerve cell adhesion molecule-L1 (L1CAM), nerve cell adhesion molecule-1 (NCAM-1), and other proteins involved in nerve development are highly correlated with PHH in premature infants . Clinicap < 0.05)  . The CSF < 0.05) . It is sIn conclusion, the authors found that CSF-TP is closely related to PHH. Because the laboratory parameters of CSF are difficult to obtain continuously in a single patient, a large amount of data is needed for analysis. A total of 156 samples that met these requirements were collected. Time was an important factor in PHH, and change in CSF-TP was found to be time-dependent. Although the CSF-TP level decreased after 2 weeks, some of the patients still developed PHH. Therefore, CSF-TP × time was innovatively analyzed as an index, and after setting the reference value at CSF-TP × time ≥ 9,600  and pooling the data into groups, the multi-factor analysis showed that it is an independent risk factor. Taking the average CSF-TP value every 2 days to establish a linear analysis, it was found that CSF-TP in the PHH group gradually decreased to normal levels within 18 days and lasted for ~35 days, and that CSF-TP in the NPHH group was significantly lower. Therefore, we suspected that patients in the PHH group developed PHH within 18 days. CSF-TP was divided into three groups , and the proportion of complicated PHH every 4 days was observed: <400 mg/L group samples were too less to obtain statistically significant data, and nine cases in the >16-day group had complicated PHH. Finally, it was observed that the proportion of PHH in the 400–1,200 mg/L and >1,200 mg/L groups gradually increased with time, with a more obvious increasing trend for the latter.There are some limitations to this study. First, this was a retrospective, single-center study, which may have led to unclear data collection and information deviation. Second, other factors that may have affected the results cannot be excluded. Therefore, more clinical data are required, and further prospective randomized trials are needed to confirm the findings of this study.Posthemorrhagic hydrocephalus (PHH) is a common complication of neurosurgical hemorrhagic brain disease. There is a lack of research on the pathogenesis and risk factors of CSF-TP. In this study, the clinical data were analyzed using univariate and multivariate analyses, and it was found that GCS score <8 and CSF-TP × time  were important risk factors for PHH, and that CSF-TP in the PHH group was higher than in the NPHH group. Lastly, the time course of changes in CSF-TP levels significantly differed between the two groups.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Biological and Medical Ethics Committee of Jiangxi Provincial People's Hospital (No. 2021-074). Written informed consent for participation was not required for this study in accordance with the national legislation and institutional requirements.RL and HX conceived and supported this research and revised the manuscript. ZW, YC, and XZ designed and conducted the present study, and drafted the manuscript. ZW and FM conducted statistical analysis and interpreted the data. YC and CW collected the primary data. FM and CW completed the follow-up and collected the associated data. ZW and XZ participated in the statistical analysis of data and article writing. All authors of this study met the ICMJE criteria for authorship, made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, and drafting, critical revision, and final approval of this manuscript.This study was supported by the Beijing Municipal Science and Technology Commission (Grant No: Z181100001518005).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients often present with CSF rhinorrhea, recurrent meningitis, chronic headache, and visual defects, while few patients have been reported to present with neuroendocrine alterations. Endonasal endoscopic repair is the first-line treatment for these leaks at present. However, the relatively high risk of recurrence remains the main cause of reoperation because of elevated intracranial pressure (ICP) after endoscopic surgery and absence of postoperative ICP management. A shunting procedure may stop CSF leakage or relieve symptoms in complex cases, and this is presently well-known as the last-line therapy for CSF liquorrhea. We describe a 29-year-old woman with spontaneous CSF rhinorrhea and neuroendocrine alterations due to IIH, and with no previous history of trauma, tumor, or nasal surgery. The bone defect in the skull base became implicated when the site of the leak was detected by cranial magnetic resonance imaging and computed tomography (CT). The patient was successfully managed  Spontaneous cerebrospinal fluid (CSF) leaks due to idiopathic intracranial hypertension (IIH) are a relatively uncommon but serious disease. Patients generally manifest CSF liquor fistula, which leads to serious consequences such as meningitis, chronic headache, and visual defects, while few patients have been reported to present with neuroendocrine alterations . Endoscovia ventriculoperitoneal shunt (VPS) alone, without endoscopic endonasal skull base surgery.This study presents an exceedingly rare clinical presentation of spontaneous CSF rhinorrhea, irregular menstruation, and infertility in an adult female patient who had elevated ICP. The patient was successfully managed 2 who was previously healthy presented with complaints of intermittent watery discharge from the right nostril for 4 years when bowing her head, irregular menstruation, and infertility for 3 years. Her past medical and surgical history was unremarkable, and she had no history of head injury. Furthermore, no abnormalities were found in the physical examination, and no abnormalities were found during the ultrasound examination of the heart, liver, kidneys, uterus, or ovaries. Beta-2 transferrin was identified in the fluid outflow by biochemical analysis. Furthermore, the serum level of prolactin (PRL) was elevated (100 ng/ml). All other laboratory values for pituitary hormone were normal. No drugs were used to induce hyperprolactinemia, such as antiepileptic or antipsychotic drugs. The quantitative value of the ICP measured by lumbar puncture before treatment was 190 mmH2O.A 29-year-old woman with a body mass index (BMI) of 35 kg/mThe bone defect in the body of the sphenoid sinus implicated at the site of the leak was detected in the axial CT scan . The cor2O. Then, the patient was followed up for 2 years after hospital discharge. The patient recovered well postoperatively with a normal PRL level in serum, had no clinical or radiological evidence of recurrence, and became pregnant twice. Furthermore, the sagittal T2 images revealed that no CSF accumulated in the sphenoid sinus  findings of IIH, (2) bony defect(s) and herniating contents, and (3) potential risk factors, such as pterygoid recess pneumatization, arachnoid pits, or defects . To dateThe present case had imaging findings of IIH with enlarged lateral ventricles and third ventricle, and had evidence of elevated CSF pressure. The excellent CT scanning through the skull base identified bone defects in the body of the sphenoid sinus under optic chiasma, which was implicated at the site of CSF leak. The sagittal T2 images revealed that the fluid accumulated in the sphenoid sinus with a gas-liquid plane, and the coronal T2 images revealed the presence of arachnoid pits along the anteromedial aspect of the middle cranial fossa. No extended or embedded sac was found in the sphenoidal sinus.via medication or shunting procedure can be an important adjuvant treatment in this patient population (P < 0.001) in patients undergoing surgical repair of CSF leakage  for the publication of any potentially identifiable images or data included in this article.GL contributed to the conception of the study. DP performed the investigation and data curation. KY modified the grammar mistakes. CW and WS contributed significantly to the analysis and manuscript preparation. FG helped to perform the analysis with constructive discussions. All authors contributed to the article and approved the submitted version.This work was supported by the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (2020372207).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cerebrospinal fluid (CSF) leak can be spontaneous or nonspontaneous. The management options include conservative treatments, blood patch, and surgical repairs. We compared clinical symptoms, image findings, management options, hospitalization, and relapse rates among different causes of CSF leaks. Eighty-one patients were recruited: 20 with spontaneous and 61 with nonspontaneous CSF leaks. Nonspontaneous causes included lumbar puncture, surgery, and trauma. Surgery sites comprised sphenoid, spine, skull base, and calvaria. Spontaneous CSF leak came from the sphenoid or spine. Age, gender, body mass index, initial symptoms, hospitalization, treatment courses, and recurrence rates showed no difference between the groups. The spontaneous group had higher CSF accumulations on their MRIs. MRI pachymeninge enhancement showed the highest sensitivity (78.6%) for intracranial hypotension. Meningitis occurred in 1/3 of sphenoid, skull base, and calvarian surgeries. Earlier reoperation was correlated with shorter hospitalization (r = 0.651), but the recurrence rates were similar. Longer intervals between surgery and CSF leak encouraged reoperation. Among the spontaneous spine and lumbar puncture-related CSF leaks, 57.1% of them responded to 4 days of conservative treatment. Among the trauma-related CSF leaks, 90.9% of them required surgical repair. The demographic data and symptoms were similar in various groups of CSF leak. The symptom onset durations and treatment strategies were different. However, the recurrence rates were similar. Cerebrospinal fluid (CSF) leak can be spontaneous (primary) or nonspontaneous (secondary) . ClinicaThe International Classification of Headache Disorders, 3rd edition [We reviewed medical charts from August 2004 to August 2021, and recruited patients diagnosed with CSF leak and headache attributed to low CSF pressure based on  edition ,10.The conservative treatment protocol included intravenous hydration, optional caffeine intake, and strict bed rest without head elevation for over 23 h a day . Eating,Clinical data, imaging findings, treatment options, duration of hospitalization, and symptom recurrence were collected and analyzed. Onset duration was defined as the time period from the day of symptom onset to the day of seeking medical assistance. Conservative treatment duration refers to the time span from the day of hospitalization to the day of first invasive management or discharge. Hospitalization duration lasted from admission until discharge; for those who had CSF leak after a procedure during the same hospitalization, the hospitalization duration was counted from the day of symptom onset to discharge. Days before recurrence were calculated from the day of the last successful treatment to the day of symptom recurrence.t test was used for continuous variables. The chi-square test was utilized for categorical data; if the data number was fewer than 5, Fisher’s exact was applied. Ordinal data, such as the visual analogue scale for pain, were analyzed with the Mann–Whitney test. Statistical significance was defined as p < 0.05. All statistical analyses were carried out using R (version 4.1.1) for Windows.The two-tailed Student’s MRI findings of intracranial hypotension were defined as enhancement of the pachymeninges or any two of the following: subdural fluid accumulation, engorgement of venous structures, pituitary hyperemia, and sagging of the brain ,13. EachThis study was approved by the E-DA Hospital Institutional Review Board (IRB)—EMRP-110-115.The International Classification of Headache Disorders, 3rd edition. Twenty-one patients had accidental CSF leak during surgery but did not have symptoms postoperatively, and all of them were excluded. We further excluded three patients with tumor invasion of the skull bone and two patients with osteomyelitis. Eight-one patients entered the final analysis . We divided patients into two groups based on the spontaneity of leak . The spoNonspontaneous CSF leak was caused by surgery, lumbar puncture, and trauma . Based oIn surgery-related CSF leak that required advanced surgical treatment, further analysis showed that the surgeons’ decisions for reoperation were associated with longer intervals from the last operation to CSF leak but not with symptom onset duration or pain severity A. Once CTrauma-related CSF leak was predominantly in males  at the age of 34.5 ± 10.6 years old. Symptoms occurred 2 ± 1.7 days after trauma and rhinorrhea was more common than headache as the presenting symptom. Image findings of CSF collecting in the anterior skull base were found in 54.5% of the patients.Spontaneous CSF leak was subdivided anatomically into sphenoid, cervical, thoracic, and lumbar regions based on the leaking sites . The unkSeventy-nine percent of patients with spontaneous spinal CSF leak had MRI findings of both intracranial hypotension and 71.4% showed CSF accumulation. MRI findings of intracranial hypotension had an overall sensitivity of 85.7%. With detailed analyses, enhancement of the pachymeninges showed the highest sensitivity of 78.6%, followed by subdural fluid accumulation and sagging of the brain, both 21.4%, and subsequently by engorgement of venous structures and pituitary hyperemia, both 14.3%. We arrived at a new finding that enhancement of the pachymeninges was not necessary to involve the whole of the meninges. One patient had involvement of the frontotemporal lobe and the other had involvement of the occipital area only. Both were confirmed with low intracranial pressure by lumbar puncture .p = 0.462), MRI signs of intracranial hypotension (p = 0.070), symptom onset durations (p = 0.058), or pain scale (p = 0.085). Interestingly, among the patients treated with blood patch, two had recurrences, and among those treated supportively, three had recurrences. The recurrence rates were similar (p = 1).We further analyzed the results of blood patch on spinal CSF leak, including the spontaneous spinal CSF leak group and the lumbar puncture subgroup of the nonspontaneous CSF leak patients. There were 9 patients treated with blood patch and 12 patients treated supportively. Patients treated with blood patch had shorter conservative treatment durations of 4 ± 1.9 days, compared with the 7.3 ± 4.4 days in the conservative treatment group; however, the total hospitalization showed no difference C. Thus, This is the first systematic evaluation of the management and assessment of the outcomes of all causes of CSF leak. Previous studies mainly focused on incidence or prevention of procedure-related CSF leak. In surgery-related CSF leak, we excluded 21 patients of incidental durotomy during surgery without clinical symptoms. A previous study on spinal surgery showed that incidental durotomy was found in 3.84% of all spinal surgeries and only 1/4 of them had clinical symptoms . This exSpinal surgery-related CSF leak had a prevalence from 4.7% to 14% at the lumbar spine and 1% at the cervical spine. With regard to surgical procedures, cervical anterior decompression had the lowest incidence (0.42%) of CSF leak, and lumbar decompression ± fusion without instrumentation had the highest rate 17%) . Among o% . AmongMeningitis was a common complication during the treatment. There was no difference in the prevalence of meningitis between the spontaneous and nonspontaneous groups; however, subgroup analysis of nonspontaneous CSF leak showed higher meningitis complication rates in sphenoid, skull base, and calvarian surgeries, compared with spinal surgery, trauma, and lumbar puncture. This suggests that surgeries involving the skull have a higher chance of meningitis complication.Our findings showed that earlier reoperation would shorten the hospital stay. However, surgeons preferred conservative treatment unless there was a longer interval between the last operation and CSF leak. We further analyzed the recurrence rates of surgical and conservative treatment groups and found that there was no significant difference. Earlier reoperation could shorten the hospital stay but could not reduce the recurrence. Reoperation, of course, cost more than the conservative treatment did.Sphenoid and spinal CSF leak were both considered as spontaneous CSF leak but they were fundamentally different. All cases of sphenoid CSF leak presented with rhinorrhea but not headache, which was similar to previous reports . SphenoiIn spontaneous CSF leak of the spine, although our case number was not large, the demographic data were similar to those previously reported with female predominance and onset age in the forties. MRI was reported to be diagnostic of approximately 80% of the cases, which is also similar to our findings at 85% . Our datWe had a strict definition of trauma-related CSF leak, which was not complicated with intracranial hemorrhage and needed to be severe enough to cause clinical symptoms. Young male with rhinorrhea was the most common presenting symptom; 81.8% required surgical treatments. A recent large-scale review of CSF leak following skull-base trauma had a similar demographic data to ours . We had This study has several limitations. The case number of each subgroup was relatively small, which imposed statistical limitations on detailed subgroups analyses. We tried to determine the demographic and treatment variances in different group of patients; however, multiple comparisons further impact on the significance. This is a retrospective study and some data were not documented universally, such as the time span from the uprightness to the headache onset, the history of connective tissue disorder, and the CSF pressure. Ex post facto analysis was thus restricted. We did not have data on the total number of operations performed in each subgroup and thus the actual incidence and prevalence could not be calculated. We considered sphenoid and spinal leak together as the spontaneous CSF leak and conducted the analysis. They are significantly different in structural, pathologic, etiological, and treatment perspective. In spontaneous spinal and lumbar puncture-related CSF leak, 42.9% of the patients were referred for blood patch after conservative treatment for 4 ± 1.9 days. Thus, we conclude that, after an average of 4-day conservative treatments, 57.1% of these patients showed good responses. However, further prospective study is required to confirm our presumption.We conducted a retrospective study on all causes of CSF leak. Interestingly, although patient groups were previously considered different, there were no differences in demographic data, initial symptoms, days of hospitalization, and even recurrence rates between spontaneous and nonspontaneous CSF leak. In surgery-related CSF leak, calvarian surgery needed three repetitive operations while other surgeries required just one operation. Meningitis was more common in surgery involving the skull and did not happen in spinal surgery or lumbar puncture-related CSF leak. In spontaneous CSF leak, leak from the sphenoid and from the spine showed a difference in initial symptoms and ways of management, while MRI findings and the recurrence rate were similar. Enhancement of the pachymeninges was most sensitive for intracranial hypotension on MRI. For the management of surgery-related CSF leak, earlier surgery repair was associated with a shorter hospital stay, but only longer intervals between surgery and CSF leak encouraged surgeons to reoperate. We advised that trauma-related CSF leak be treated aggressively. Spontaneous CSF leak had a 57.1% response rate under the 4-day supportive treatment. Blood patch was reserved for more severe conditions but did not shorten or prolong hospitalization.