Herpes zoster is characterized by the presence of unilateral, localized vesicular rash in a dermatomal distribution. Although in most cases herpes zoster affects a single dermatome, multidermatomal involvement may occur in rare cases.In this case report, we describe an immunocompetent patient with multidermatomal herpes zoster involving the cranial nerve (CN) V3 and C2 territories with simultaneous vestibulocochlear deficits. We demonstrate the results of temporal bone magnetic resonance imaging and discuss the pathophysiological mechanism of the patient’s characteristic clinical manifestation.The diagnosis of multidermatomal herpes zoster involving CN V3 and C2 territories with simultaneous vestibulocochlear deficit was made. Vestibular function test and pure tone audiometry revealed ipsilateral vestibular impairment and hearing loss.The patient was admitted for treatment with systemic steroids and antiviral agents.After treatment, vertigo and hearing loss improved, and the skin lesions healed well without scarrings.Considering that the afferent sensory fibers of the CN V and C2 converge in the trigeminocervical nucleus, herpes zoster involving contiguous dermatomes of CN V3 and C2 in the patient may be explained by neural-to-neural spread. And considering that temporal bone magnetic resonance imaging revealed enhancement of the dura of the internal auditory canal without enhancement of CN VII or VIII, the vestibulocochlear deficits in the patient may be caused by inflammation of the inner ear transmitted via the cerebrospinal fluid. Herpesviridae. VZV infection is species-specific to humans. Herpes zoster infection is caused by reactivation of VZV in the cell bodies of neurons. Initial infection with VZV is called varicella or chickenpox and usually occurs in childhood; after the initial infection, a latent infection is established in the cranial nerve ganglia or sensory dorsal root. Under some conditions, the dormant VZV reactivates and migrates along the nerve axons to the skin, causing vesicular eruptions that show a localized dermatomal distribution. Herpes zoster generally manifests as painful vesicular eruptions along the unilateral distribution of the dermatome, most commonly affecting a single thoracic truncal dermatome. Less commonly, involvement of the cervical dermatome or cranial nerves (CNs), especially CN V and CN VII, has been reported.–3 Herpes zoster oticus (HZO) refers to VZV reactivation within CN VII, and is characterized by an erythematous vesicular rash in the external ear and otalgia. When accompanied by ipsilesional facial weakness, a diagnosis of Ramsay Hunt syndrome can be made. In HZO, vestibulocochlear symptoms such as hearing loss, tinnitus, and vertigo are often present, and the pathological mechanism of vestibulocochlear deficit has been suggested to be directional spread of VZV to CN VIII from CN VII or CN VIII via the cerebrospinal fluid.–8Varicella zoster virus (VZV), also known as human herpesvirus 3, is a double-stranded DNA virus belonging to the family Here, we present a 35-year-old woman with herpes zoster in multiple adjacent dermatomes involving CN V3 and C2, concomitant with peripheral-type vertigo and hearing loss on the ipsilateral side.http://links.lww.com/MD-CASES/A23). The direction of nystagmus did not change when the head position was changed. No abnormal findings were observed on neurological examinations such as the cerebellar function test and lower cranial nerve examination. Facial weakness was not observed on either side. A bithermal caloric test showed right unilateral weakness with a canal paresis value of 78% , and written informed consent was obtained from the patient for publication of this case report details.This case presentation raises 2 interesting issues that are worth studying. First, herpes zoster involving multiple adjacent dermatomes is very uncommon in young, healthy, immunocompetent patients. Second, except in cases of Ramsay Hunt syndrome, the concomitant vestibulocochlear deficit on the ipsilateral side without evidence of CN VII involvement is also very unusual in individuals with herpes zoster. In some patients, herpes zoster extends beyond a single dermatome, and this multidermatomal involvement often occurs in the presence of underlying immunocompromised conditions such as human immunodeficiency virus infection, hematological malignancy and chemotherapy. Multidermatomal herpes zoster involving the trigeminal nerve,–12 or cervical or thoracic dermatomes has rarely been reported in immunocompetent patients. The exact mechanism underlying multidermatomal herpes zoster has not been clearly elucidated. Yoon et al reported a case of left Ramsay Hunt syndrome with extensive dissemination of vesicular eruptions to the bilateral scalp, posterior neck, shoulder, upper arm, and posterior upper neck in an immunocompetent elderly patient. The authors assumed that the widespread dissemination of herpes zoster infection may occur through interconnections between ganglia or due to viremia of VZV. Gomez and Chernev reported a 95-year-old patient with disseminated multidermatomal herpes zoster involving the left CN VI-III, the bilateral trunk and bilateral extremities and presented a review of 28 immunocompetent patients with disseminated herpes zoster previously reported in the literature. The results showed that the initial presentation of vesicular rash most frequently occurred in dermatomes innervated by the cranial nerves (42%), followed by the thoracic (27%), lumbosacral (19%), and cervical (15%) dermatomes. Extracutaneous manifestations were reported in twelve cases (43%); among theses, mucosal lesions, central nervous system lesions, cranial or peripheral nerve paresis, Ramsay Hunt syndrome, herpes ophthalmicus, and visceral involvement were observed in 4, 2, 2, 2, 1, and 1 cases, respectively. The authors speculated that widespread dissemination of herpes zoster may be caused by VZV viremia, and this was supported by the previous finding that VZV DNA can be amplified from the serum of immunocompetent patients with localized herpes zoster. Our patient presented with characteristic skin findings of herpes zoster involving contiguous dermatomes of CN V3 and C2. It has been reported that the afferent sensory fibers of the CN V and C2 converge in the trigeminocervical nucleus, and VZV spread between these nerves may occur at this point. Another plausible explanation would be spreading of the virus from skin to skin or from peripheral nerve to peripheral nerve, considering that the dermatomal territories show abundant overlap in head and neck region.Herpes zoster generally develops unilaterally in a single dermatome. The thoracic dermatomes are most commonly affected, followed by the cervical and trigeminal dermatomes.,19,20 we suspected herpes zoster oticus as a cause of the vestibulocochlear deficits in this patient. In our previous studies, vestibulocochlear deficits were observed in more than half of patients with Ramsay Hunt syndrome, and postcontrast temporal bone MRI in these patients exhibited enhancement of inner ear structures and of the dura of the internal auditory canal as well as of the facial nerve and the external auditory canal.–8,21,22 However, our patient did not show facial weakness or enhancement of the facial nerve or the external auditory canal, suggesting that the vestibulocochlear deficits were not accompanied by Ramsay Hunt syndrome or herpes zoster oticus. Concomitant vestibulocochlear deficits and ipsilateral multidermatomal herpes zoster involving the CN V3 and C2 territories in the absence of Ramsay Hunt syndrome has not previously been reported in the literature, and the mechanism of this clinical manifestation is not clear. Although anastomosis between the auriculotemporal branch of CN V3 and the temporofacial division of CN VII has been demonstrated, there is no anastomosis between CN V3 and CN VIII. Thus, in our patient, the possibility of VZV spread via neural anastomoses or interganglionic transmission may be low. When VZV infection extends to the cerebrospinal fluid, central nervous system infections such as encephalitis, encephalomyelitis or meningitis have been reported.–25 Eskiizmir et al described an immunocompetent 19-year-old man with Ramsay Hunt syndrome and simultaneous VZV encephalitis. Previously healthy patients with disseminated cutaneous zoster and aseptic meningitis have also been reported.,25 Considering that no evidence of meningitis was found in our patient and that temporal bone MRI revealed enhancement of the dura of the internal auditory canal without enhancement of CN VII or VIII, the vestibulocochlear deficits in our patient may be caused by inflammation of the inner ear transmitted via the cerebrospinal fluid.Another noteworthy manifestation in our patient was comorbid hearing loss and vertigo due to ipsilateral vestibulocochlear deficit. Because Ramsay Hunt syndrome can be associated with herpes zoster involving CN V or C2, as reported in previous studies,To our knowledge, this is the first case report of multidermatomal herpes zoster involving the CN V3 and C2 territories with simultaneous vestibulocochlear deficits. The mechanism of herpes zoster involving contiguous dermatomes of CN V3 and C2 may be neural-to-neural spread, and that of vestibulocochlear deficit may be viral inflammation of the inner ear transmitted via cerebrospinal fluid.Conceptualization: Chang-Hee Kim.Data curation: Taesik Jung, ChanEui Hong, Jung Eun Shin, Chang-Hee Kim.Formal analysis: Taesik Jung, Jung Eun Shin, Chang-Hee Kim.Funding acquisition: Chang-Hee Kim.Investigation: Taesik Jung, ChanEui Hong, Jung Eun Shin, Chang-Hee Kim.Methodology: Taesik Jung, ChanEui Hong, Chang-Hee Kim.Supervision: Chang-Hee Kim.Visualization: Jung Eun Shin.Writing – original draft: Taesik Jung, Chang-Hee Kim.Writing – review & editing: Chang-Hee Kim.


Mucormycosis is a fatal fungal infection, and there is limited information on its epidemiology and treatment practices, including the optimal dosage of liposomal amphotericin B.A retrospective nationwide analysis of 82 proven and probable cases of mucormycosis was performed. Cases were collected from 51 hospitals in Japan by hematologists and infectious disease specialists. Variables included annual incidence, site of infection, Mucorales genera, and patient and treatment characteristics. Comparison was made between patients who survived and those who died. Comparative analysis was performed between patients who received a liposomal amphotericin B dose of 5 mg/kg and those who received a dose of >5 mg/kg. Categorical variables were tested for differences using Fisher's exact test or χ-squared test. The association between liposomal amphotericin B dose and prognosis was evaluated using the Kaplan-Meier method, and differences were tested using the log-rank test. A Cox proportional hazards model was used to control for confounding.The lung was the most commonly involved organ (70.7%), and 35.4% of cases were disseminated. Rhizopus spp., Cunninghamella spp., and Mucor spp. were the most common organisms. No significant differences in infected organs or distribution of Mucorales were observed between the survival and death groups. Longer duration of neutropenia was more common among deaths (p = 0.007). Mortality within 120 days was 54.2%. More cases in the survival group started liposomal amphotericin B before confirmed diagnosis (p = 0.028). Survival did not differ between patients receiving liposomal amphotericin B at 5 mg/kg and those receiving >5 mg/kg (p=0.873). Using Cox proportional hazards models adjusting for confounders, such as age ( >65 years), prolonged neutropenia, and resection of infected lesions, the hazard ratio for the effect of >5 mg/kg liposomal amphotericin B on 120-day prognosis was 0.93  compared with 5 mg/kg.This study provides important insights into the precise epidemiology and treatment practices of mucormycosis among hematologists and infectious disease specialists. There was no difference in prognosis between the 5 mg/kg and >5 mg/kg liposomal amphotericin B groups.Masato Tashiro, MD, PhD, Asahi Kasei Pharma Corporation: Advisor/Consultant|Asahi Kasei Pharma Corporation: Honoraria|Sumitomo Pharma Co., Ltd.: Honoraria Hiroshi Kakeya, MD, PhD, Asahi Kasei Pharma Corporation: Honoraria|Merck Sharp & Dohme: Honoraria|Sumitomo Pharma Co., Ltd.: Honoraria Hiroshige Mikamo, M.D, Ph.D, Asahi Kasei Pharma Corporation: Grant/Research Support|Merck Sharp & Dohme: Honoraria|Pfizer Inc.: Grant/Research Support|Pfizer R&D Japan: Honoraria|Sumitomo Pharma Co., Ltd.: Grant/Research Support|Sumitomo Pharma Co., Ltd.: Honoraria Kazutoshi Shibuya, M.D., Ph.D., Sumitomo Pharma Co., Ltd.: Grant/Research Support Koichi Izumikawa, M.D., Ph.D., Asahi Kasei Pharma Corporation: Grant/Research Support|Asahi Kasei Pharma Corporation: Honoraria|Astellas Pharma Inc.: Honoraria|DAIICHI SANKYO COMPANY, LIMITED: Grant/Research Support|DAIICHI SANKYO COMPANY, LIMITED: Honoraria|KYORIN Pharmaceutical Co., Ltd.: Honoraria|Merck & Co., Inc.: Honoraria|Pfizer Japan Inc.: Honoraria|Shionogi & Co., Ltd.: Grant/Research Support|Shionogi & Co., Ltd.: Honoraria|Sumitomo Pharma Co., Ltd.: Grant/Research Support|Sumitomo Pharma Co., Ltd.: Honoraria|TAIHO PHARMACEUTICAL CO., LTD.: Grant/Research Support


In the Acknowledgments section, “This study did not receive funds, grant, or other support.” was incorrect. This should have read “This research was financially supported by Fondazione CRT‐Turin, Italy [grant number 73537].”We apologize for this error.


The corrected Acknowledgments are as follows.B.S. acknowledgesSRA MERGE for creating a stimulating scientificcommunity. B.S. and J.R. acknowledge FORMAS for financial support.C.P. acknowledges the Lund Institute of Technology for postdoc funding.J.R. and A.C.E. further acknowledge financial support via Grant AEROMETII 19ENV08. N.L.P. and J.J.L. acknowledge funding from the Academyof Finland  andthe European Research Council (ERC) under the European Union’sHorizon 2020 Research and Innovation program .


Intracochlear schwannomas (ICS) are very rare benign tumours of the inner ear. We present histopathological proof of the extremely rare bilateral occurrence of intracochlear schwannomas with negative blood genetic testing for neurofibromatosis type 2 (NF2). Bilateral schwannomas are typically associated with the condition NF2 and this case is presumed to have either mosaicism for NF2 or sporadic development of bilateral tumours. For progressive bilateral tumour growth and associated profound hearing loss, surgical intervention via partial cochleoectomy, tumour removal, preservation of the modiolus, and simultaneous cochlear implantation with lateral wall electrode carrier with basal double electrode contacts was performed. The right side was operated on first with a 14-month gap between each side. The hearing in aided speech recognition for consonant-nucleus-consonant (CNC) phonemes in quiet improved from 57% to 83% 12 months after bilateral cochlear implantation (CI). Bilateral intracochlear schwannomas in non-NF2 patients are extremely rare but should be considered in cases of progressive bilateral hearing loss. Successful tumour removal and cochlear implantation utilizing a lateral wall electrode is possible and can achieve good hearing outcomes. They arma ILS; –4). Thes. ThesinnWith an incidence of 0.81 per 100,000 person-years, IES are a very rare subtype of schwannomas of the eighth cranial nerve . They weintracochlear schwannomas (ICS). Patients with ICS can present with hearing loss (in 94.5% of cases), more rarely sudden hearing loss (29.6%), tinnitus (67.6%), and vertigo (50.7%) [The most common subsite for IES is intracochlear and these tumours are therefore known as ing loss 9.6%, tininnitus 6.6%, and  (50.7%)  describeCochlear implantation (CI) with penetration of the ICS and no tumour removal has also been described in the literature , 11. ComWithers et al. (2020) previously published an extremely rare case of bilateral ICS established on clinical and radiological MRI findings . Here weandA 63-year-old Caucasian female patient initially presented with an 8‑year history of right-sided hearing loss, associated with non-pulsatile tinnitus and two brief episodes of vertigo. The MRI examination demonstrated an enhancing small mass within the cochlea, suggestive of a right ICS. A “wait-and-scan” approach was initially decided with close surveillance. On subsequent serial imaging an early-stage left-sided ICS was also identified Fig.  with botA blind sac closure of the external auditory meatus with obliteration of the eustachian tube and canal wall-down mastoidectomy was performed prior to tumour resection with a microscope. On the right side the ICS was identified on opening the round window with tumour extending to the cochlea–carotid junction of the basal turn Fig. . The lefNF2 gene mutation in unaffected tissues (blood) using multiplex ligation-dependent probe amplification  and SRY-related HMG-box 10 protein (sox-10), but was negative for epithelial membrane antigen (EMA) and anticytokeratin monoclonal antibodies AE1 and AE3 (AE1/3). This was consistent with a benign schwannoma for both sides Figs.  and 6. An .Fig. 5RAt the routine 8‑week post-operative review, following her second procedure , the patient had well-healed surgical sites. She was able to perform well with gait, Rhomberg and Unterberg testing. Formal balance testing showed mild right beating nystagmus with oculomotor testing with Frenzel goggles. She had positive bilateral head impulse and negative Hallpike and log roll testing. Her imbalance subjectively improved with nil balance concerns 6 months after the second procedure. Post-operative testing showed good electrode impedance with no open or short circuits on each side. At the last post-operative review, she had been wearing the left cochlear implant for 16 months and the right one for 24 months.Aided speech perception (CNC words in quiet for phonemes delivered at 65 dB sound pressure level) indicated excellent hearing bilaterally and a significant binaural benefit Table . The lefDue to susceptibility artefacts from bilateral cochlear implants, MRI of the middle ear and membranous labyrinth was not able to assess tumour recurrence; however, macroscopic  gross tumour clearance without remnant tumour was achieved at the time of surgery. The patient continues to receive routine audiological follow-up and implant assessment.Intracochlear schwannomas confined exclusively to the cochlea are very rare tumours, let alone occurring bilaterally. This case is—to the best of our knowledge—the first of its kind in the published literature confirming the presence of ICS on histopathology, immunohistochemistry as well as genetic testing. A case published by Nam et al. (2011) revealed bilateral intracochlear schwannomas on post-mortem temporal bone histopathology in NF2 patients . HoweverWhile bilateral ICS in patients with no genetic or clinical features of NF2 appear to be extremely rare, the possible explanations for this include mosaicism for NF2 or sporadic development of bilateral tumour development by chance alone . PreviouOnce diagnosed, many factors contribute to the management of ICS, including tumour size, location and growth, initial hearing status and deterioration as well as patient factors. Especially patients with serviceable hearing and without vertigo can be monitored by surveillance  similarly to the management of “classic” VS in the IAC or CPA. However, tumour growth into the vestibule or through the modiolus into the internal auditory canal complicates management, because hearing rehabilitation with a cochlear implant and complete tumour removal is not possible in transmodiolar tumours and vertigo attacks in the patient’s history decrease the chance of preservation of vestibular function after subtotal cochleoectomy , 26. ForSince the possibility of hearing rehabilitation after radiation of an intracochlear tumour is unclear, this may be considered for tumours which have invaded critical areas including IAC or cerebellopontine angle.Improved surgical visibility was achieved in the described case following a blind sac closure and partial cochleoectomy. This was advantageous for sufficient surgical view with the surgical microscope, both for tumour removal and CI placement. Other approaches including combined tympanomastoid and maintaining the external ear canal have been described –29. The Good audiological outcomes have been obtained with ICS tumour removal despite partial or even subtotal cochleoectomy . PreservBilateral intracochlear schwannoma are extremely rare but may occur in non-NF2 patients.Patients presenting with such bilateral schwannomas later in life may not have NF2, with the likelihood of mosaicism or chance occurrence present.Simultaneous cochlear implant insertion following tumour removal can be performed with successful hearing rehabilitation even when utilizing a lateral wall electrode.


Background: Systemic anticoagulation with heparin during cardiopulmonary bypass (CPB) should be neutralized by protamine administration to restore normal hemostasis. Our previous study showed the protamine-to-heparin ratio (P-to-H) of 1:1  is likely an overestimation. Thus, we reduced the P-to-H in the HMS Plus Hemostasis Management System to 0.9:1 (0.9 Ratio) for 5 months and then to 0.8:1 (0.8 Ratio). We monitored post-operative (post-op) bleeding in the setting of reduced protamine dose (PD). Methods: We performed a retrospective study of 632 patients  who underwent cardiac surgery to measure the reduction of PD and how it affects 24-hour (24 h) post-op chest tube output. We also analyzed the entire data set to explore whether further reduction of P-to-H is warranted. Results: While there was no difference in the indexed heparin dose among the three groups, we achieved a significant reduction in the indexed actual protamine dose (APDi) by 24% (0.9 Ratio) and 31% (0.8 Ratio) reductions compared to the 1.0 Ratio group. On average, APDi was 88 ± 22, 67 ± 18, and 61 ± 15 mg/m2 in the 1.0, 0.9, and 0.8 Ratio groups, respectively. We found no significant difference in 24 h post-op bleeding among the three groups. Conclusion: 1.0 Ratio at the completion of CPB is likely an excessive administration of protamine. With the stepwise reduction of PD, we observed no increase in post-op bleeding, which may indicate that no meaningful increase in heparin rebound occurred. In addition, further analysis of the entire data set demonstrates that a 0.75 Ratio is likely sufficient to neutralize the heparin completely. At the completion of cardiopulmonary bypass (CPB), an optimal amount of protamine should be administered to restore normal hemostasis from systemic anticoagulation with heparin. With the administration of an optimal protamine dose (PD), neither residual heparin nor excessive protamine would be present. It is believed that protamine underdosing may cause heparin rebound to increase post-operative (post-op) bleeding –4 while There is no consensus on how to determine the optimal PD. One of the most common strategies is a fixed protamine-to-heparin ratio (P-to-H) with either the first heparin bolus (FHB) or total heparin dose (THD) during CPB, which does not account for heparin metabolism during CPB. Other strategies are developed to consider heparin metabolism and/or heparin concentration (HC) such as mathematical calculations of PD either using statistical or pharmacokinetic modeling of heparin metabolism over time or as a function of baseline and post-heparin ACTs; PD based on a measured HC, which should improve the accuracy of heparin neutralization and reduce PD , 19–25.In general, it is considered that 1 mg protamine is sufficient to neutralize 100 international units (IU) of heparin (1.0 Ratio) , 26. HowWe reviewed the anesthesia and perfusion records of 313 patients from 1/1/2018 to 4/5/2018 who had cardiac surgery with CPB at Massachusetts General Hospital (MGH) with P-to-H of HMS being set at 1:1 (1.0 Ratio), 296 patients from 3/11/2020 to 7/15/2020 at 0.9:1 (0.9 Ratio), and 302 patients from 8/24/2020 to 11/25/2020 at 0.8:1 (0.8 Ratio). Patients with the following criteria were excluded for accurate analysis: missing or incomplete data and patients with a heart transplant, lung transplant, LVAD insertion, red blood cell transfusion between heparin dose response test (HDR) and FHB or deceased. We excluded 104, 85, and 95 patients, resulting in 209, 211, and 212 patients to be analyzed in the 1.0, 0.9, and 0.8 Ratio groups, respectively.in vitro using the HMS to determine baseline ACT (bACT), and individual slope and calculated heparin concentration (CHC) to reach target ACT (tACT) before or right after induction of anesthesia. Our HMS is set to use IU of heparin and we used porcine heparin sodium (1000 USP units/mL) from SAGENT Pharmaceuticals  or FRESENIUS KABI . Our tACT on CPB is ≥400 s and target HC (tHC) is ≥2.0 IU/mL. Our practice at MGH to determine FHB to go on CPB is described in detail elsewhere [We performed an HDR test lsewhere –29. AddiTo determine the PD required to neutralize heparin at the completion of CPB, we measure the HC 5–10 min after removal of the cross-clamp. P-to-H is expressed as either mg protamine:100 IU heparin or a Ratio such as 1:1 or 1.0 Ratio throughout this manuscript. P-to-H in our HMS was set at 1:1 (1 mg protamine per 100 IU heparin), 0.9:1, or 0.8:1 as indicated for the calculation of PD. HMS calculates and provides three protamine doses: patient, pump, and total protamine doses. The calculated total protamine dose (CTP) is the calculated pump dose plus the calculated patient protamine dose (CPP).Our guideline recommends using CPP based on our previous study . Howeverp; Student’s t-test, p), and the box and whisker plots. We employed heparin and protamine doses indexed to a body surface area (BSA) such as indexed FHB (FHBi), indexed THD (THDi), and indexed APD (APDi) for objective comparisons among three different groups. We calculated BSA and TBV following Du Bois and Du Bois, 1916 [We used Microsoft Office Excel 365  to perform data input, calculations, statistical analysis  of the circulating heparin at the completion of CPB is likely an overestimation  reduced significantly from 152 ± 53 (1.0 Ratio) to 133 ± 46 (0.9 Ratio) and 116 ± 37 mg (0.8 Ratio), which is reflected in the actual protamine dose (APD) of 174 ± 53 (1.0 ratio) to 136 ± 46 (0.9 Ratio) and 121 ± 36 mg  than bACT, possibly indicating errors in the HPT test (data not shown). This data is essentially identical to the 1.0 Ratio group where four patients had residual heparin but two of the 4 had lower ppACT than bACT  and 31% (0.8 Ratio) reductions in APDi compared to the 1.0 Ratio group . On averan bACT  can be used as a reliable indicator to analyze a safe minimum protamine dose, 80–90% of ppACT/bACT is a good indicator of proper neutralization of heparin and 94% of CPP on average can be a safe minimum protamine dose to neutralize heparin at the completion of CPB .We combined all three groups and categorized them into APD/CPP distribution to find the smallest possible P-to-H. All CPPs are recalculated in P-to-H of 1:1 for this analysis. As expected with three groups combined, the averages of APD/CPP are widely distributed as 75–141% of CPP . HoweverAdequate neutralization of heparin by protamine at the completion of CPB is important to minimize bleeding after CPB. Although protamine is an effective means of reversing heparin, adverse events, including hypotension, pulmonary edema, and anaphylaxis, can occur. In addition, free protamine with excessive doses has been linked to increased post-op bleeding. Administering an optimal dose of protamine to neutralize heparin completely without the excessive free protamine should help to minimize these adverse events while ensuring hemostasis , 32–33.Since there is no available measurement of free circulating protamine, protamine dosing has been largely empirical. It is usually expressed as protamine: heparin ratio. A common recommendation is a fixed 1:1 (1 mg protamine to every 100 IU of heparin) or 0.8 ratios based on FHB needed to establish therapeutic anticoagulation to go on CPB. Other strategies are based on THD during CPB or the latest circulating HC. Thus, it is important to clarify what the base of the ratio is to discuss the protamine to heparin ratio , 19–25.2). This translates to, on average, our P-to-H was 0.32:1 for THD and 0.46:1 for FHB, which is significantly lower than the conventional fixed ratio of 1:1 or 0.8:1 of FHB  , 4 and hOur study shows that, in the discussion of heparin and protamine doses of two or more independent groups, it is important to use the doses indexed to BSA since a larger total blood volume is expected with the larger BSA. This is shown in our data that the 0.9 group has a larger BSA than the 1.0 and 0.8 groups, which is statistically significant. This resulted in a significant increase in FHB in the 0.9 compared to the 1.0 and 0.8 Ratio groups. However, there was essentially no change in the FHBi and the first HC 3 min after FHB , which sIn addition, further analysis of our entire data set shows that there is more room to reduce P-to-H to 0.75 mg protamine to 100 IU circulating heparin to completely neutralize the circulating heparin , which iLimitations: PD can be dependent on the tACT on CPB. Our tACT on CPB is 400 s and tHC is 2.0 IU/mL or higher –29. It i


The complex interplay between chemical, electrical, and mechanical factors is fundamental to the function and homeostasis of the brain, but the effect of electrochemical gradients on brain interstitial fluid flow, solute transport, and clearance remains poorly quantified. Here, via in-silico experiments based on biophysical modeling, we estimate water movement across astrocyte cell membranes, within astrocyte networks, and within the extracellular space (ECS) induced by neuronal activity, and quantify the relative role of different forces  on transport and fluid flow under such conditions. We find that neuronal activity alone may induce intracellular fluid velocities in astrocyte networks of up to 14μm/min, and fluid velocities in the ECS of similar magnitude. These velocities are dominated by an osmotic contribution in the intracellular compartment; without it, the estimated fluid velocities drop by a factor of ×34–45. Further, the compartmental fluid flow has a pronounced effect on transport: advection accelerates ionic transport within astrocytic networks by a factor of ×1–5 compared to diffusion alone. Over the last decades, the neuroscience community has paid increased attention to the astrocytes—star-shaped brain cells providing structural and functional support for neurons. Astrocyte networks are likely to be a crucial pathway for fluid flow through brain tissue, which is essential for the brain’s volume homeostasis and waste clearance. However, numerous questions related to the role of osmotic pressures and astrocytic membrane properties remain unanswered. There are also substantial gaps in our understanding of the driving forces underlying fluid flow through brain tissue. Answering these questions requires a better understanding of the interplay between electrical, chemical, and mechanical forces in brain tissue. Due to the complex nature of this interplay and experimental limitations, computational modeling can be a critical tool. Here, we present a high fidelity computational model of an astrocyte network and the extracellular space. The model predicts the evolution in time and distribution in space of intra- and extracellular volumes, ion concentrations, electrical potentials, and hydrostatic pressures following neural activity. Our findings show that neural activity induces strongly coupled chemical-mechanical-electrical interactions in the tissue and suggest that chemical gradients inside astrocyte syncytia strengthen fluid flow at the microscale. The complex interplay between chemical, electrical, and mechanical factors is fundamental to central nervous system physiology –3 and paThe renewed interest in brain clearance pathways over the last decade, supported by breakthroughs in imaging , experimThese questions seem intrinsically related to interactions between mechanical and chemical forces, and molecular diffusion juxtaposed with electrical and advective drift, but have largely not been addressed as such. The focus in computational neuroscience has conventionally been on electrophysiology alone , predomiIn Halnes et al. , the autHere, our target is two-fold: we aim to estimate the water movement induced by neuronal activity across astrocyte cell membranes, within astrocyte networks, and within the extracellular space, and to determine the relative role of different forces  on ionic transport and fluid flow under such conditions. To estimate this electro-chemo-mechanical response, we introduce a high-fidelity computational model describing the spatial and temporal dynamics at the micro/milliscale of volume fractions, electrical potentials, ion concentrations, and hydrostatic pressures in an intracellular space (ICS) representing different astrocyte configurations and the extracellular space (ECS) . The modOur findings show that neuronal activity in the form of extracellular ionic input fluxes induces complex and strongly-coupled chemical-electrical-mechanical interactions in the astrocytic ICS and ECS. The response is characterized by membrane (electric) potential depolarization on the order of tens of millivolts and ECS water potentials ranging from a few to a hundred kilo-pascals, spatial differences in osmolarity on the order of several tens of millimolars, and fluid velocities ranging from a fraction—to tens of micrometers per minute. The fluid dynamics are crucially coupled to the spatial organization of the intracellular network. We observe intracellular fluid velocities in astrocyte networks of up to 14 μm/min, and fluid velocities in the ECS of similar magnitude. These velocities are dominated by an osmotic contribution in the intracellular compartment; without it, the estimated fluid velocities drop by a factor of ×34–45. Furthermore, the compartmental fluid flow has a pronounced effect on transport: advection accelerates ionic transport within astrocytic networks by a factor of ×1–5 compared to diffusion alone.In order to quantify the relative role of osmotic, hydrostatic, and electrical forces on transport and flow in cortical tissue, we ask the following questions. How do astrocyte and extracellular ion concentrations, electric potentials, pressures, and interstitial fluid velocities respond to changes in extracellular ion concentrations mirroring neural activity on the time scale of seconds? Moreover, to what extent do the mechanical responses  contribute to alleviating ionic and mechanical ECS distress? To address these questions, we introduce a set of biophysical models for these quantities of interest, governed by the balance of mass, momentum, and charge, in combination with astrocyte membrane mechanisms in a representative volume.αr, the ion concentrations [Na+]r, [K+]r, and [Cl−]r, the electrical potential ϕr, and the hydrostatic pressure pr in both ICS (r = i) and ECS (r = e). Ionic transport is driven by diffusion, electric drift, and advection. To model fluid movement in each compartment, we consider three different model scenarios:Ion- and fluid movement in an astrocyte network (ICS) and the extracellular space (ECS) is modeled via coupled partial differential equations (PDEs) in a homogenized model domain , MethodsM1 The intra- and extracellular fluid flow is driven by hydrostatic pressure gradients. The astrocytic compartment can be interpreted as either a single closed cell or as a syncytium of cells without intercellular osmotic flow.M2 The intracellular fluid flow is driven by osmotic and hydrostatic pressure gradients, and the extracellular fluid flow is driven by the same mechanism as in M1. The astrocytic compartment can be interpreted as a syncytium of cells where osmosis acts as a driving force for fluid flow.M3 The intracellular fluid flow model is the same as in M2, and the extracellular fluid flow is driven by electro-osmosis in addition to hydrostatic pressure gradients. To include electro-osmosis as a driving force for ECS fluid flow is motivated by the narrowness of the ECS e, a decrease of 18.7 mM in [Na+]e, and a decrease of 16.9 mM in [Cl−]e, measured at the center of the input zone . Taking the difference in solute potential across the membrane gives us the osmotic pressure, which differs substantially between the models: M0, M1, and M2 predict a maximum drop in osmotic pressure of respectively 121 kPa, 0.713 kPa, and 20.2 kPa , the ECS solute potential increases . The incThe presence of driving forces for fluid flow—both at the brain microscale and organ-level—remain an open question. To assess the potential contributions from osmosis in the astrocytic network (M2) and electro-osmosis in the ECS (M3), we here compare the compartmental fluid velocities, cellular swelling, and hydrostatic pressures predicted by model scenarios M1, M2, and M3.μm/min—about 45 times larger than for M1  and the advection/drift fraction (Fdrift) for each of the ionic species  (+ flux is 70 μmol/(m2s)  is maximum 59 μmol/(m2s), whereas for M3, the charge flux is maximum 71 μmol/(m2s).We observe that ransport . In modeto 0.949 . For K+ ol/(m2s) , whereasol/(m2s) . For Na+ic drift . The advt = 10 s and t = 210 s for all protocols and same amplitude for all input currents  input fluxes. To study how low-frequency waves of ionic changes affect the osmotic, hydrostatic, and electrical response of the system, we compare three different stimulus protocols via model scenario M3: (I) (slow) input fluxes varying at a low (1Hz) frequency, (II) (ultraslow) input fluxes varying at an ultralow (0.05 Hz) frequency, and (III) constant input fluxes, with on- and offset times + concentration, and ECS volume fraction reach periodic steady states for both the slow and ultraslow stimuli  for the slow and ultraslow stimuli, respectively, with the corresponding numbers being 60% and 53% for ϕe and 74% and 63% for αe  to 1 ⋅ 10−12 m2/(Pa s) ), the superficial fluid velocity increases from 0.0 μm/min to 7.1 μm/min for M1 and from 0.0 μm/min to 15 μm/min for M3 (κ = 1.4 ⋅ 10−13 m2/(Pas), while the superficial fluid velocity in M1 continues to increase through κ = 1 ⋅ 10−12 m2/(Pas). The difference between M1 and M3 in superficial fluid velocities decreases for κ values above the default parameter choice, but we still observe a notable difference of 215% at κ = 1 ⋅ 10−12 m2/(Pas). For κ values below the default parameter choice, the absolute difference between M1 and M3 fluid velocities decreases, but the relative difference increases. Increasing Km from 0.0 Pa to 3000 Pa  increases the superficial fluid velocity linearly from 0.0 μm/min to 0.40 μm/min for M1  to 1.628 ⋅ 10−13m/(Pas) ), the superficial fluid velocity increases from 0.0 μm/min to 0.32 μm/min for M1 and from 0.0 μm/min to 14 μm/min for M3  for ηm as low as 5.43 ⋅ 10−15m/(Pas). To study how changes in the input flux density −7mol/(m2s) for M1 and 9.05 ⋅ 10−7mol/(m2s) for M3). Both modeling setups show a linear relationship between By increasing n for M3 . The supn for M1 . For M3,n for M3 . The diflocities . The abs+ influx in conjunction with Na+ efflux, reflecting a zone of high neuronal activity, induces a strongly coupled and complex chemical-electrical-mechanical response in astrocyte ICS and the ECS with spatial and temporal changes in osmolarities, swelling, electrical potentials, pressures, and fluid flow. Cellular swelling and, importantly, osmotically driven fluid flow within the astrocytic network contribute to preventing high levels of extracellular water potential, effectively protecting against neuronal swelling. Fluid flow within each compartment may reach tens of μm/min and, as such, substantially contributes to the overall dynamics. Compartmental fluid flow, in concert with cellular swelling, alleviates osmotic pressure build-up and accelerates ionic transport within astrocytic networks by a factor of ×1–5 compared to diffusion alone.Our results demonstrate that localized extracellular K+ is measured to increase by 6—12 mM during sensory stimulation and strong electrical stimulation r, [K+]r, and [Cl−]r, the electrical potentials ϕr, and the hydrostatic pressures pr. The model is embedded in the electrodiffusive Kirchhoff-Nernst-Planck framework and builds on previous work on ionic electrodiffusion i threshold, and PKe (mol/m3) is the [K+]e threshold.We adopt the ionic membrane mechanisms from Halnes et al. 2013 . The mecL1, L2] with L1 = 1.35 ⋅ 10−4 m and L2 = 1.65 ⋅ 10−4 m, during the time interval . The neurons are not modeled explicitly, but we mimic their activity by injecting a K+ current into the ECS and removing the same amount of Na+ ions simultaneously. The input flux jin (mol/(m2s)) is constant. The constant stimulus is used for all simulations shown throughout this paper, except for the simulations illustrated by dark green and blue lines in Figs To stimulate the system, we follow the same procedure as in Halnes et al. 2013 . We assu+ ions from the extracellular space at a given decay rate and add the same amount of Na+ ions. The decay is proportional to the extracellular K+ concentration and defined across the whole domain. Specifically, the decay current 2s)) is given by:kdec denotes the [K+]e decay factor, and [K+]e,init the initial extracellular K+ concentration.To mimic neuronal pumps and cotransporters, we remove KAll parameters of the system are as listed in E of individual astrocytes in the hippocampus for different deformation frequencies with E ≈ 300 Pa for 30 Hz, E ≈ 420 Pa for 100 Hz, and E ≈ 520 Pa for 200 Hz, and a Poisson’s ratio ν = 0.47. We use the mean Young’s modulus (413 Pa) to calculate the bulk modulus (stiffness constant) Km as:Lu et al. 2006  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 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The work is motivated by recent interest in motion of interstitial fluid and cerebrospinal fluid, which has implications for edema, stroke, vascular dementia, and neurodegenerative disease. The ions considered include K+, Na+, and Cl-. The astrocyte wall is modeled as elastic, allowing cellular expansion and contraction. A set of ten coupled partial differential equations describes the system and is solved numerically in a time-dependent, one-dimensional domain. Neuronal activity is simulated by injecting K+ and Na+ near the center of the domain. A grid convergence study is presented. Four scenarios are studied: one in which fluid velocity is assumed to be zero (dubbed M0), one in which fluid velocity is driven by hydrostatic pressure gradients alone (M1), one in which hydrostatic pressure effects are retained and are complemented inside astrocytes by osmotic pressure gradients (M2), and one in which both of those effects are retained and are complemented outside astrocytes by electro-osmosis (M3). The resulting concentrations increase and decrease with curves of the sort we might expect  and have values of the same order of magnitude as found in prior experiments and simulations. In scenario M1, the authors find a two-roll fluid circulation into astrocytes at the location of ion injection, along cell bodies, and out again at the domain edges. They also find that osmotic pressure differences between astrocytes and extracellular spaces are small in scenario M2 but larger, and generally similar, in scenarios M1 and M3. Fluid velocities are similar in scenarios M2 and M3. A sensitivity analysis shows that varying the compartmental permeability, membrane stiffness, and membrane permeability over reasonable ranges alters maximum transport rates relatively little.The modeling and manuscript are thorough and meticulous. The results are clear enough and extend significantly beyond prior studies, which have not accounted for coupling among so many mechanisms. The interpretations are straightforward and not overwrought. Thus I strongly support publication, providing the authors respond effectively to a few questions and comments:Figure 2 shows temporal variations that look like smooth exponential rise and decay in every case except one, the concentration of K+ ions (panel D). What is happening there?The results consistently show that osmotic pressures are much higher than hydrostatic pressures. How much error would be introduced if hydrostatic pressures were neglected altogether?The scenarios simulated all consider quasi-steady behaviors between step changes in neuronal activity . Of course, many other timescales are of interest neurologically. Most of all I wonder what the model would predict for slow / ultraslow frequencies of the sort considered by Fultz et al. (Ref. 1). Could the authors run the model with neuronal activity varying at 0.05 Hz or 1 Hz? Or at least speculate in the Discussion about what might occur?Finally I noticed (just two) typos:line 387 - acknowledges should be acknowledgeline 153 - stabilize should be stabilizingReviewer #2: Sætra ploscompbio2023Interest in the nature and drivers of fluid flow within the brain has taken on renewed interest with the idea that this movement may be important for clearance of waste from the brain through the glymphatic system. Because these flows are technically difficult to measure, they have been the focus of many computational models, though most of them have focused on mechanical drivers . This paper investigates the intra and extracellular fluid flows induced by ionic/osmotic currents  in simple model of the astrocyte syncytia and extracellular space. The authors find that there is fluid flow away from the site of activity and into the astrocytes remote from the site. The authors find that including osmotic and electrodiffusion in their models have a large effect on flow. There has been very little work done in this area and this looks to be an important contribution.Major comments:The 6.7mM K+ external change that is used here is very high, well beyond the standard physiological range. Increases in vivo are more in the range of 1 mM  for some measurements on naturalistic conditions). That being said, looking at the equations I do not see anything that would prevent the flows scaling linearly (or something like that) with linear increases in injected /withdrawn K+/Na+. Can the authors make address this point, either analytically or with simulations if this is the case?Will multiple areas of activity lead to a linear superposition of the observed dynamics?The abstract and summary are vague and do not contain concise descriptions of the results. They should be re-written so that what is shown in the paper is summarized in the abstract.I think the paper would be strengthened by exploring a different set of boundary conditions. The no-flow conditions here are fine as a simplification, but in the real system one might expect a constant pressure/ionic concentration boundary that might produce different responses, particularly if the activity location is moving. The “holy grail” of all this glymphatic system modeling is to get directed fluid flow (left to right or vice versa in this model’s extracellular space). Is there any way the authors could generate something like this, or suggest experiments that could test their ideas?Minor comments:Don’t use dotted lines- it makes it hard to see sharp transitions.A zoom in on the onset offset dynamics in figure 2 would be helpful for the reader to understand the timescale of the changes here.Mark default values on figure 7.ReferencesRasmussen R, Nicholas E, Petersen NC, Dietz AG, Xu Q, Sun Q, Nedergaard M (2019) Cortex-wide Changes in Extracellular Potassium Ions Parallel Brain State Transitions in Awake Behaving Mice. 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Read more information on sharing protocols at  8 Jun 2023AttachmentResponse to reviewers.pdfSubmitted filename: Click here for additional data file. 28 Jun 2023Dear Dr. Sætra,We are pleased to inform you that your manuscript 'Neural activity induces strongly coupled electro-chemo-mechanical interactions and fluid flow in astrocyte networks and extracellular space - a computational study' has been provisionally accepted for publication in PLOS Computational Biology.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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I encourage publication.Reviewer #2: The authors have addressed all my concerns.**********Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********what does this mean?). 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The US has experienced a significant decrease in CLABSI rates over time but is stalling. NHSN has rigid definitions for secondary BSI attribution. We reviewed CLABSIs in a large tertiary acute care hospital to determine the frequency and trends of potential misattribution.We reviewed 282 CLABSIs cases from 2019 to 2023 at a large tertiary hospital. A team of infection preventionists with high inter-rate reliability adjudicated each case strictly following NHSN methodology. A clinical review was performed by two hospital ID-trained epidemiologists to explore misattributed with an alternative primary infection.Of 282 CLABSI cases, 65% occurred in adults and 35% in children. 122 cases (43%) had a potential primary infection. The potential primary infections included: 33 (27%) pneumonia, 28 (23%) gastrointestinal infections, 6 (5%) urinary infections, 4 (3%) surgical infections, 13 (11%) skin and soft tissue infections, 11 (9%) vascular infections, 5 (4%) bacterial translocation, 8 (6.5%) infections that were present on admission, 4 (3%) CNS infections, 2 (1.6%) NEC infections, 2 (1.6%) musculoskeletal, 2 (1.6%) contaminates and 4 (3%) other non-NHSN specified sites. Pneumonia was the most frequent misattribution failing to meet imaging, laboratory, or documentation requirements within the infection window period (IWP). Intra-abdominal infection was the second most frequent cause of misattribution due to not meeting the imaging or matching site culture within the IWP. For the other categories, the most frequent reasons for not meeting the primary criteria included lack of matching culture, lack of clinical symptoms (often in intubated patients), or in the endocarditis category, the complexity of the criteria even with positive blood cultures and presence of vegetations. The graph shows an increasing proportion of misclassification over time.Percent of Misattributed CLABSI by Fiscal YearNHSN definitions and rules are becoming too prescriptive and may no longer be applicable to the complexity of cases, overestimating CLABSIs. As hospitals commit to CLABSI reduction, accurate and specific definitions are key in focusing efforts and attention and avoiding unfair penalties.Luis Ostrosky-Zeichner, MD, FACP, FIDSA, FSHEA, FECMM, CMQ, Astellas: Grant/Research Support|Cidara: Advisor/Consultant|Cidara: Advisor/Consultant|F2G: Advisor/Consultant|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|NIH: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: Honoraria|Pulmocide: Grant/Research Support|Scynexis: Grant/Research Support|T2 Biosystems: Grant/Research Support|Viracor: Advisor/Consultant


Contemporary data reflecting local pathogens and their antibiograms is necessary to select empirical antimicrobial therapy for equine neonates.Describe bacterial isolates associated with equine neonatal infection and their antibiograms in the Midwestern United States. An increase in gram‐positive infection and antibiotic resistance compared to previous literature was expected.Data from 149 fluid samples from 133 foals <30 days of age submitted for bacterial culture between January 2007 and December 2018.A retrospective evaluation of equine neonatal fluid cultures was performed. Fluid submission type, bacterial culture and antibiogram, empirical antibiotic treatment, and foal outcome was included. Isolate susceptibility to individual antimicrobials and combination protocols relevant to equine practice were recorded. The effect of recorded variables on foal survival was evaluated using Fisher's exact or chi‐squared tests.Ninety bacterial isolates (78 aerobes and 12 anaerobes) were identified and gram‐positive organisms predominated . Greater than 70% of aerobic isolates were susceptible to ampicillin, ceftiofur, chloramphenicol, trimethoprim/sulfamethoxazole, and all penicillin/aminopenicillin and aminoglycoside combinations. Seventy‐seven (n = 81/105) percent of foals survived. Survival was associated with a negative fluid culture and was not associated with empirical antimicrobial choice.Gram positive and anaerobic isolates associated with equine neonatal fluid cultures exceed that of previous reports. Historical empirical antimicrobial choices for equine neonatal infection in the Midwestern United States are supported by local antibiogram results. CLSIClinical and Laboratory Standards InstituteMDRmultiple drug resistanceMICminimum inhibitory concentration1Equine neonatal sepsis is the combination of demonstrable infection and clinical evidence of systemic inflammation which includes at least 3 of the following: elevated rectal temperature, tachycardia, tachypnea, abnormal leukocyte count , venous hyperlactatemia, or venous hypoglycemia.2Bacterial fluid cultures including blood, synovial, cerebrospinal, and peritoneal samples from foals <30 days of age submitted to the Michigan State University Veterinary Diagnostic Lab from January 2007 to December 2018 were reviewed. Field and hospital acquired samples were included. When multiple fluid cultures were submitted from a single foal, each culture was evaluated independently and comparisons between results described.Hospital submitted blood cultures were collected aseptically at the time of intravenous jugular catheter placement. Briefly, 7 to 8 mL of blood was sterilely collected and used to inoculate commercially available aerobic  and anaerobic  fluid cultures. Hospital submitted nonblood cultures were sterilely collected and the sample was either inoculated into fluid culture broth as above or placed in a vacutainer tube without additive based on sample volume. Aerobic and anaerobic media inoculation and culture submission are routine for the reporting hospital; however, aerobic culture was prioritized if sample volume was small. Sample collection technique was not available for field submissions. The percentage of submissions with inoculation of both aerobic and anaerobic media was not determined.In vitro susceptibility to antimicrobials was recorded for aerobic isolates using broth microdilution method. Briefly, 10 μL of 0.5 McFarland suspension of the bacteria in sterile water was inoculated to 10 mL of cation‐adjusted Mueller‐Hinton broth. Fifty microliters of this bacterial suspension were added to each well of an equine specific antimicrobial sensitivity testing plate  and the plate was incubated at 35°C for 16 to 18 hours. After incubation the minimum inhibitory concentration (MIC) for each antimicrobial was manually read and recorded. Antimicrobials were included for analysis if they were considered practical for equine neonatal use and susceptibility data was reported for at least 50% of all bacterial isolates obtained. Amikacin, ampicillin, cefazolin, ceftiofur, chloramphenicol, enrofloxacin, gentamicin, penicillin, tetracycline, and trimethoprim/sulfamethoxazole were included. Bacterial susceptibility was also reported for antimicrobial combinations common to equine practice and included amikacin/ampicillin, amikacin/penicillin, gentamicin/ampicillin, and gentamicin/penicillin. Susceptibility data was reported in ordinance with Clinical and Laboratory Standards Institute (CLSI) established breakpoints. Multiple drug resistant (MDR) isolates were defined by resistance to 3 or more evaluated antimicrobials.Case data including age, breed, and sex were recorded for all foals, and antimicrobial therapy and survival data were reported for all hospitalized foals. Empirical antimicrobial therapy was defined as the first antimicrobial administered to the foal. This therapy was categorized as “appropriate” if the antimicrobial demonstrated in vitro efficacy against organism(s) isolated from the foal's first fluid culture submission(s). Empirical antimicrobial efficacy was only evaluated if: the initial fluid sample yielded positive growth, an antibiogram was available for the recovered isolate(s) and the specific empirical antimicrobial used was recorded. If serial fluid cultures were submitted, changes in the isolated bacterial agents and their antibiograms were reported but not used to define empirical antimicrobial efficacy. Survival was defined as survival to hospital discharge and was not considered for field cases. Clinical and biochemical case data required to identify systemic inflammation was not evaluated, and enrolled foals with positive fluid cultures could not be defined as septic.Foal age was tested for normality using a Shapiro test, and year of submission was assessed for uniform distribution. Categorical data included submission type , fluid type, bacterial isolate, antimicrobial sensitivity, empirical antimicrobial efficacy, and survival. Relationships between these categorical variables were evaluated using a Fisher's exact (2 variables) or chi‐squared (>2 variables) tests.3One hundred forty‐nine fluid cultures were performed on 133 foals <30 days of age. This included 124 hospital acquired fluid cultures  and 25 field acquired fluid cultures . Seventy‐six males and 49 females were enrolled and sex was not recorded in 8 foals. Breeds were representative of the local population and included quarter horse/paint, other/mixed, Arabian, warmblood, standardbred, and thoroughbred most commonly.P = .02). Sixty‐six percent (n = 98/149) of fluid submissions were blood and 34% (n = 50/149) were synovial fluid; 1 cerebrospinal fluid culture was submitted. Synovial fluid samples were overrepresented in field submissions (P = .0001). Fluid culture submissions were uniform across the study time period.Average age at the time of fluid culture submission was 6.6 days , and age at submission differed between blood and synovial fluid samples  from 63  foals yielded 93 bacterial organisms. Of these organisms 3 could not be accurately identified, leaving 90 isolates for analysis  followed by Actinobacillus spp. , Staphylococcus spp. , Streptococcus sp.  and Enterococcus sp. . Table Seventy‐eight  aerobic and 12  anaerobic organisms were isolated, and gram‐positive organisms predominated . Specifically, 50% (n = 39/78) of aerobes and 92% (n = 11/12) of anaerobes were gram positive , multiple synovial fluid cultures (n = 2) repeat blood cultures (n = 1) and blood and CSF cultures (n = 1). For no foal were all isolates identical in repeat samples; for 6 foals the same bacteria were isolated from multiple cultures, but in each of these 6 foals disparate isolates were also recovered.3.1X2  = 6, P = .11; gram positive: X2  = 1.6, P = .65; gram negative: X2  = 5.4, P = .14; Table Susceptibility profiles for all were available for 78% of aerobic isolates (n = 61/78), and included 67% n = 26/39) of gram‐positive and 90% (n = 35/39) of gram‐negative aerobes. Chloramphenicol was the most effective in vitro antimicrobial across all aerobes, followed by ceftiofur. Ninety‐two percent (n = 56/61) of all aerobes were susceptible to chloramphenicol including 96% (n = 25/26) of gram‐positive and 89% (n = 31/35) of gram‐negative aerobes. Eighty‐five percent (n = 52/61) of all aerobes were susceptible to ceftiofur, including 73% (n = 19/26) of gram‐positive and 94% (n = 33/35) of gram‐negative aerobes. Greater than or equal to 70% of bacterial isolates were susceptible to ampicillin , trimethoprim/sulfamethoxazole , and all combination protocols , Streptococcus spp. , Enterobacter spp. , Salmonella sp. , Actinobacillus sp. , Citrobacter freundii , E. coli  and Staphylococcus xylosus . Fifty percent (n = 13/26) of gram‐positive and 22% (n = 8/35) of gram‐negative aerobes were MDR. Greater than 70% of MDR isolates were sensitive to chloramphenicol  and the combination of amikacin/ampicillin  but all combination protocols remained statistically noninferior .Thirty‐four percent (n = 20/61) of isolates demonstrated resistance to >3 relevant antimicrobials and were considered MDR. This included 3.2P = .05). Of the 63 foals with positive initial fluid culture results, empirical antimicrobial therapy was not reported for 8 field cases, and complete antibiogram data for recovered isolates was not reported for 30 hospitalized cases which most commonly this occurred when an anaerobe of laboratory‐defined contaminant organism was isolated.Survival data was available for 105 of 108 hospital admitted foals and was unavailable for all foals evaluated in the field. Seventy‐seven percent (n = 81/105) of foals survived to hospital discharge. Foals with growth of at least 1 bacterial organism from at least 1 fluid culture were less likely to survive  compared to foals with negative fluid cultures . All foals for which empirical antimicrobial choice was not appropriate had MDR bacteria isolated from blood culture.As a result, empirical antimicrobial therapy and complete antibiogram data was available for only 25 foals with positive initial fluid culture results and 64% (n = 16/25) of these foals survived. Empirical initial antimicrobial therapy was considered appropriate based on subsequent antibiogram data in 75% (n = 19/25) of foals. Four of the remaining 6 foals received ceftiofur sodium at extra‐label doses (5‐10 mg/kg IV Q6‐12). Sixty‐eight percent of foals that received appropriate initial antimicrobials (n = 13/19) and 50% of foals that did not receive appropriate empirical antimicrobials (n = 3/6) survived , exceeding that of previous reports (1.2%‐6.3%).Fluid submission type was associated with isolate gram status. Gram‐negative isolates were more common in synovial samples, and gram‐positive isolates were more common in blood samples. However, this difference was largely attributed to gram‐positive anaerobes, and their recovery be related to systematic differences in the categories of fluid collected in this study. Firstly, collected synovial fluid volume might be insufficient to inoculate both anaerobic and aerobic media, with preference given to aerobic inoculation. Secondly, synovial fluid samples were overrepresented in field submissions and anaerobic media inoculation of foal fluid samples might not be routine for field practitioners, although this was not quantified. It is, however, also possible that there are pathophysiologic differences in infection based on gram status in foals as is seen in human medicineUnique bacterial isolates were cultured from different fluid samples in 7/10 foals. This could be a consequence of sample contamination, or might reflect the dynamic nature of bacterial infection in foals. A recent study comparing serial blood cultures in equine neonates found that approximately 20% of foals had novel bacterial growth after ≥48 hours of hospitalization compared to admission,Greater than 70% of recovered isolates were susceptible to ampicillin, ceftiofur, chloramphenicol, trimethoprim sulfamethoxazole, and all antimicrobial combination protocols. There were no significant differences in susceptibility across combination protocols. Chloramphenicol was the most effective individual antimicrobial, with 92% of isolated bacteria demonstrating in vitro sensitivity. However, its use in equine neonates faces pharmacologic and practical limitations. In adult horses repeat oral dosing of chloramphenicol fails to achieve CLSI MIC targets for >50% of the conventional dosing interval.In this study, 85% of isolates were susceptible to ceftiofur at the CLSI MIC breakpoint of ≤2 μg/mL. This exceeds isolate susceptibility in geographically distinct studies.Actinobacillus spp. were susceptible to combination protocols containing penicillin, whereas 100% were susceptible to protocols containing ampicillin. Sixty‐nine percent of gram‐negative isolates were susceptible to gentamicin and 75% to amikacin, supportive of emerging aminoglycoside resistance.Despite increased isolate sensitivity to ceftiofur compared to contemporary reports, other antibiogram results of this study support emerging trends in antimicrobial resistance.Seventy‐seven percent of foals survived to hospital discharge and survival was associated with a negative fluid culture as reported.This study faces several limitations related to both sample size, retrospective data collection, and inclusion of both hospital and field‐based submissions. Incomplete and small data sets prevented some useful observations, such as comparing culture and antibiogram results across the study period, or between hospital and field submissions, which are reported.Fluid cultures are inherently susceptible to both false negative and false positive results, which might under‐ or overestimate the prevalence of clinically relevant isolates. Until more precise bacteriologic identification methods are routinely available, all reports of equine neonatal infection are liable to these inaccuracies. Polymicrobial infection or disparate serial fluid culture results reported in this study might represent sample contamination; however, both findings are also reported clinicallyAuthors declare no conflict of interest.Ceftiofur sodium used off‐label.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.


Brain tumours have significant impacts on patients’ quality of life, and current treatments have limited effectiveness. To improve understanding of tumour development and explore new therapies, researchers rely on experimental models. However, reproducing tumour-associated epilepsy (TAE) in these models has been challenging. Existing models vary from cell lines to in vivo studies, but in vivo models are resource-intensive and often fail to mimic crucial features like seizures. In this study, we developed a technique in which normal rat organotypic brain tissue is implanted with an aggressive brain tumour. This method produces a focal invasive lesion that preserves neural responsiveness and exhibits epileptiform hyperexcitability. It allows for real-time imaging of tumour growth and invasion for up to four weeks and microvolume fluid sampling analysis of different regions, including the tumour, brain parenchyma, and peritumoral areas. The tumour cells expand and infiltrate the organotypic slice, resembling in vivo behaviour. Spontaneous seizure-like events occur in the tumour slice preparation and can be induced with stimulation or high extracellular potassium. Furthermore, we assess extracellular fluid composition in various regions of interest. This technique enables live cell confocal microscopy to record real-time tumour invasion properties, whilst maintaining neural excitability, generating field potentials, and epileptiform discharges, and provides a versatile preparation for the study of major clinical problems of tumour-associated epilepsy. Given the high mortality and morbidity of brain tumours and the lack of effective treatments, a variety of model systems have been developed, from in vitro tumour cell lines to in vivo animal models. A major goal of brain tumour research is to find a model that adequately recapitulates tumour-tissue interactions, as well as more complex effects such as the development of tumour-associated epilepsy, which occurs in a large proportion of subjects with considerable morbidity ,2. CurreAs an alternative to in vivo approaches, we developed an in vitro model in which tumour cells with a well-defined lineage are implanted focally into rat organotypic brain slices . This haThe methodology of the in vitro brain tumour model described has several steps, which are outlined graphically in All procedures were carried out in accordance with protocols approved by the University of Melbourne Animal Ethics Committee.v/v): 48%v/v Minimum Essential Media (MEM) + GlutaMAX , 24%v/v Earle’s Balanced Salt Solution (EBSS) without added calcium, magnesium and phenol red , 24 heat-inactivated horse serum , 2%v/v of 32.5% w/v glucose dissolved in MEM, 2%v/v B27 supplement , and 2%v/v penicillin–streptomycin . All solutions were sterilised using 0.22 μm syringe filters . Slices were incubated at 37 °C with 5% CO2, 95% O2 gas, and the slice culture media was changed 24 h after harvest and every 48 h thereafter.Six- to eight-day-old Wistar rats were rendered unconscious with isoflurane before decapitation and extraction of the intact brain into Gey’s Balanced Salt Solution (GBSS) supplemented with an additional 1 g/L of glucose. Hippocampal sections (350 µm thick) were cut with a McIlwain tissue chopper . Slices were cultured on 30 mm diameter tissue culture inserts  as described by De Simoni and Yu 2006) [ [6]. It v/v) 10%v/v foetal bovine serum , 1%v/v penicillin–streptomycin  and 1%v/v GlutaMAX  and incubated at 37 °C with 5% CO2, 95% O2.SMA-560 cells, a spontaneous murine astrocytoma cell line , stably Tumour cells were injected into brain slices 3–5 days after preparation to allow adequate time for slice stabilisation and confirmation of viability.6 cells/mL for injection into brain slices.SMA-560 cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) at a density of 2 × 10Borosilicate capillary glass  was used to make injection pipettes with a Sutter Instruments P-1000 electrode puller, after which the tip diameter was enlarged to facilitate cell delivery with gentle application of tissue paper. The final tip diameter was 300–500 μm.Cell culture well inserts containing brain slices were transferred into a 35 mm culture dish with media to allow accurate positioning of the injection pipette. The insert was kept in the incubator until the injection apparatus was set up to minimize environmental exposure.The injection pipette was inserted into the injector port of an IM-6 microinjector (Narishige) and positioned in the cell suspension. The suspension was carefully drawn up until approximately half the pipette was filled, being careful not to pull the cells into the attached tubing. A small amount of fluid was then expelled by slight positive pressure prior to positioning in the slice.The filled pipette inserted into the microinjector was attached to a Narishige MM-3 micromanipulator for positioning in the slice. It was then guided just above the surface of the slice, to the point of injection, under a dissection microscope at 4× magnification. The tip was then lowered into the slice by approximately 100 μm. The cellular suspension generally started flowing at this stage due to loss of surface tension when contact was made with the slice; this was minimised by applying slight negative pressure to inhibit flow until the desired point of injection was reached. A bolus of ~1 μL was then injected. The extent of the labelled cell injection could be checked immediately under a fluorescent microscope. The “tumour load” could be adjusted by changing the volume of injection or cell suspension density, but generally, injection volumes less than 1 μL did not survive.After injection, the insert was placed in the six-well plate and placed back in the incubator. The growth of the injected tumour mass could then be observed over several days C. The tu2, 2 CaCl2, 1.2 NaH2PO4, 23 NaHCO3, 11 D-glucose) bubbled with 5% CO2, 95% O2 gas. The present observations were made at ambient room temperature (~22 °C), but similar observations could be made at higher temperatures (32–34 °C) with a Warner TC-324C inline temperature controller. Glass pipettes for recording extracellular potentials were made from 1.5 mm thin-walled glass , with tip resistances of ~4 MΩ and filled with ACSF. A tungsten concentric bipolar microelectrode  was used for stimulation. The electrode was connected to an NPI SEC-05X amplifier and digitised at 10 kHz with an Analog devices PCI-6035E analogue to digital converter and low pass filtered at 2 kHz. A Grass SD9 stimulator provided stimulating pulses triggered from the A-D board. The software package WinWCP V5.5.6 (University of Strathclyde) was used to display and record signals.Brain slices adherent to the insert membrane were cut out and placed in a recording chamber ~2 mL volume with a coverslip bottom perfused at 1.5 mL/min with artificial cerebral spinal fluid (ACSF)  that resemble epileptic discharges and are sensitive to antiepileptic drugs, which are considered an in vitro model for evoked seizures . PADs weIn further experiments, epileptiform discharges were also evoked in both the control and tumour-bearing slices, respectively, using elevated KCl (8 mM) in the ACSF.A modification of the method of Shao and Feldman (2007) was used to sample extracellular fluid from various areas of the brain slice, including the peri-tumoural region to measure for metabolites, neurotransmitters, or other substances of interest . A glassBrain slices were preserved in 10% neutral buffered formalin (NBF) prior to staining and fixation. After paraffin embedding, 5 μm sections were cut and stained with haematoxylin and eosin for examination with light microscopy.The slices were imaged macroscopically at low power  with a non-inverted fluorescence microscope  with the culture plate cover in place. With the slices cut from the inserts, individual cells could be observed using conventional differential interference contrast (DIC) optics with a 40× water immersion lens . Additionally, it was possible to obtain more detailed observations of tumour properties, including spread into normal tissue, using a confocal laser microscope system  with a 488 nm laser and 10× Fluo/Na 0.3 objective for live cell imaging over several days. In this case, the slices were cultured on coverslips without the membrane inserts to allow visualisation of the slice and tumour growth through the bottom of the chamber.2  of about 0.1–0.3 mm2 A–C. Vari2 A,B. The 2 A–C. SlicElectrical stimulation of the Schaffer collateral pathway (SCP) resulted in typical field potentials in the CA1 region. Tetanic stimulation of the SCP resulted in primary after-potentials (PADs) and seizure-like discharges. Typical field potentials and PADs resulting from tetanic stimulation in both the tumour-injected and control slices are shown in Using this protocol, the tumour-injected organotypic hippocampal slices were found to be hyperexcitable compared with the control slices as they exhibited much higher rates of PAD events following high-frequency electrical stimulation (PAD frequency in tumour-injected slices was significantly higher G. PAD dun = 5 for each region) from parenchyma and the peri-tumoral and intra-tumoral regions in the control and tumour slices are shown in Levels of neurotransmitters and metabolites such as tryptophan, glycine, and ammonia could be measured from the microvolume fluid samples. Quantitative mass spectrometry was used to detect these constituents and quantify them using area under the curve (AOC) measurements, which allowed comparisons between control and tumour slices as well as intra-slice comparisons. AOC measurements   utilised  utiliseMore recently, the advanced in vivo and in vitro models such as the Hatcher and colleagues 2020 tumour suppressor gene deletion model  and GillThe current report further extends these studies with the use of an intra-slice, volume-adjustable injection technique producing a focal invasive tumour resembling in vivo tumours. More significantly, it extends previous methods by permitting electrophysiological observations of neural circuit activation and epileptiform activity, live cell confocal imaging, and microenvironment sampling.This model also avoids the need for genetic modification previously used in some in vivo models of brain tumour-associated epilepsy ,18,21 anIn principle, this technique may be performed with any form of fine injector such as a Hamilton syringe or even a volumetric pipette. However, the microinjector used in this study afforded excellent volume control and spillage prevention. Additionally, slices of any part of the brain could be used with this technique. The hippocampus was selected in this case due to an extensively studied structural connectivity that makes it particularly suitable for electrophysiological studies.An interesting aspect of this method was the preservation of neural transmission, as well as the ability to generate epileptiform discharges. It is anticipated that this technique will be useful for studies investigating tumour-associated epilepsy pathogenesis and treatment. Electrophysiological measurements could potentially be extended with the use of multielectrode arrays (MEAs).The ability to micro-sample extracellular fluid provides a range of further investigation. For example, it should be possible to test the “glutamate hypothesis”, which proposes that tumour-associated epilepsy is a result of excessive extracellular concentration of the potent excitatory neurotransmitter glutamic acid in the peri-tumoural brain . Further99mTc has been demonstrated to detect matrix metalloproteinases, a cell membrane-associated biomarker in glioblastomas [99mTc labelling allowed for imaging of ex vivo human brain tumour slices. The high binding specificity and ease of modification of aptamers allow for their use in future studies for the investigation of tumour-related extracellular factors of interest.Aptamers are an emerging field in cancer diagnostics and treatment using single-stranded DNA or RNA oligomers, allowing for high binding specificity to cancer biomarkers . Currentlastomas . The 99mThe organotypic-based brain tumour model technique described in this article should also allow for genetic modification of the tumour or brain environment with AAV or lentiviral vectors. This, in combination with the ability to assess tumour-related extracellular factors in the model, increases the potential complexity of this tumour model, allowing for the study of genetic driver mutations’ influence on brain tumours. The ability to image at high resolution provides another significant advantage that may be useful for studying tumour invasion processes, including invadopodia activity and modulation in real-time. The technique may also provide a platform for testing brain tumour chemotherapeutic agents and other modalities of treatment, as has been shown by Chadwick et al. (2015) , but witAlthough SMA-560 with a GFP reporter was the only tumour cell line used, it is anticipated that other lines, including human-derived glioma cell lines, would be equally suitable. However, it is particularly useful to have the option of introducing a fluorescent label. Examples of other suitable cell types that could be used in this setting include “stem cell-like” tumour lines , which aTumour cells of potentially any type may also be injected with this methodology as long as cell suspensions can be obtained. Other research areas that this technique could potentially be extended to include the exploration of metastatic tumour properties in other organs that can be cultured using the organotypic method.This organotypic-based brain tumour model demonstrates rapid tumour cell expansion and invasion into a brain tissue substrate over relatively short periods of time, as imaged with GFP fluorescence, and mimics histologically the appearance of aggressively invasive tumour cell characteristics of the SMA-560 murine astrocytoma cell line. The slices are additionally useful for electrophysiological recordings, showing preserved electrophysiological responses and epileptic-like discharges. This technique may provide an alternative to in vivo models of brain tumour growth and a simple and less resource-intensive, high throughput platform for further studies.