Daboia russelii, Echis carinatus, Hypnale hypnale, Naja naja, Bungarus caeruleus) was assessed. LC-MS/MS analysis of two batches of PAV showed the presence of 88.7–97.2% IgG and traces of other plasma proteins. The tested PAVs contained minor amounts of undigested IgG and F(ab′)2 aggregates, showed complement activation, were devoid of IgE, endotoxin, and content of preservative was below the threshold level. Immunological cross-reactivity and in vitro neutralization of enzymatic activities, pharmacological properties demonstrated superior efficacy of SL PAV compared to Indian PAV against SL snake venoms. The in vivo neutralization study showed that the tested PAVs are potent to neutralize the lethality and venom-induced toxicity of SL snake venoms. Therefore, our study suggests that introduction of SL-specific PAV will improve snakebite management in SL.Snake envenomation is a severe problem in Sri Lanka (SL) and Indian polyvalent antivenom (PAV) is mostly used for treating snakebite albeit due to geographical variation in venom composition, Indian PAV shows poor efficacy in neutralizing the lethality and toxicity of venom from the same species of snakes in SL. Therefore, the quality and in vivo venom neutralization potency of a country-specific PAV produced against the venom of the five most medically important snakes of SL ( SL is the home for 96 terrestrial and 15 aquatic species of snakes3, with the hump-nosed viper , Russell’s viper (Daboia russelii), the Indian common krait (Bungarus caeruleus), the Indian cobra (Naja naja), and the saw-scaled viper (Echis carinatus) are regarded as the most dangerous species of venomous snakes since they account for most of the snakebite fatalities in SL4.Snakebite is a severe problem in the Indian subcontinent and has been declared as a neglected tropical disease by the World Health Organization (WHO). Sri Lanka (SL), a small island of South Asia located in the Indian Ocean, reports approximately 30,000 snake envenomations that result in 450 deaths per yearD. russelii, N. naja, E. carinatus, and B. caeruleus)5. Currently, important concerns are being raised about using Indian PAV for the treatment of snakebites in SL. The primary objection is that due to geographical variations, the composition of Indian snake venoms used for immunizing and raising equine antibodies are different from the venom compositions of the same species of snakes in SL10. Consequently, Indian PAV is found to be less efficient in neutralizing the lethality and toxicity of venoms of SL snakes and additional doses of PAV are necessary for treating snakebite patients in SL. This increases the risk of adverse serum reactions in the antivenom-treated patients12 and unnecessarily increases the cost of treatment. The Indian PAVs are also limited by its absence of antibody against H. hypnale venom (HHV), which is responsible for the most envenomation in SL4. Neutralizing HHV is therefore exclusively dependent on the antibodies raised against the venom toxins of the same Viperidae family of snakes (D. russelii and E. carinatus), and consequently, the PAV that is in current use is less efficient for treating H. hypnale envenomation in SL12. Further, the supply of PAV from India is not sufficient to fulfill the annual requirements for antivenom in SL, which is causing a shortage of antivenom in this country4. These issues can be addressed by developing SL-specific PAV raised against snake venoms of SL origin, which is urgently needed for effective snakebite treatment in this country.Snakebite is a medical emergency that requires immediate medical treatment; otherwise, it may result in death and/or permanent morbidity. The only effective treatment against snakebite is intravenous administration of polyvalent antivenom (PAV). In SL, the most widely used antivenom is the Indian PAV raised against the ‘big four’ venomous snakes of India , D. russelii venom (DRV), N. naja venom (NNV), E. carinatus venom (ECV), and B. caeruleus venom (BCV) sourced from SL. Notably, the World Health Organization (WHO) Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins published in 2016, recommended that standard in vitro laboratory tests be conducted to ascertain the purity, quality, and safety of the antivenom as the essential quality control before the pre-clinical assessment of PAV15. Therefore, in addition to assessing the in vivo neutralization potency of SL snake venoms-induced lethality and toxicity by SL PAV, we also determined the physicochemical properties, purity of active substance, load of preservatives and neutralization of some enzymatic activity and pharmacological properties of SL snake venoms by this newly developed SL PAV by in vitro laboratory assessments. Our study has shown that some pharmacological properties of SL snake venoms and their enzymatic activity were neutralized by the newly developed SL PAV to a greater extent when compared to Indian PAVs that are being used for snakebite treatment in SL. The assessment indicated the enhancement of efficacy and potency of the former PAV.Recently, the pre-clinical efficacy of a newly developed SL venom-specific antivenom was reported16 but the cake-like structure of the lyophilized powder does not affect their biological activity 17. The SL PAV was found to be completely dissolved within 5–6 min in 10 mL of sterile deionized water and no insoluble material was observed. This result suggests good water solubility of lyophilized powder. The turbidity measures the cloudiness of a solution; therefore, low turbidity of a solution indicates that solute (PAV in this case) is well mixed with the solvent (sterile deionized water). Consequently, WHO has suggested for quantitative measurement of the turbidity of antivenoms solution by using a turbidimeter; albeit, no standard value of turbidity is fixed14. The turbidity of the SL PAV B1 and B2 was found to be 15.3 ± 0.6 and 11.1 ± 0.8 nephelometric turbidity units (NTUs), respectively indicating the good solubility of lyophilized powder in water. The pH of the reconstituted PAV should be around 7.0 ± 0.514. The aqueous solutions of the two batches of SL PAVs had pH values between 6.95 ± 0.2 and 6.89 ± 0.3, which is quite close to the standard set by the WHO.Examining the physicochemical properties of a drug is a preliminary but an important aspect for assessing its quality. Both vials of lyophilized PAV appeared as homogenous white colored powder, with fragment cake-like structure  without requiring refrigeration at rural hospitals. The moisture content of the lyophilized product reduces the shelf-life either by denaturing the product or by aggregating F(ab′)2 molecules that causes a significant reduction in its venom neutralization potency14. For the two batches of the tested SL PAVs, the moisture content was determined to be in the safe range (< 3%)14.In developing countries, snakebite is regarded as an occupational health hazard for rural people and a socio-economic problem13 or fragmented F(ab′)2 molecules (pepsin digested IgG to remove its Fc region)19. The Indian PAVs and the newly developed SL PAV contain F(ab′)2 and therefore theoretically believed to show less serum reactions in treated patients, however, detailed clinical studies are warranted to confirm the advantages of pepsin digested IgG20. The neutralization potency and efficacy of IgG and F(ab′)2 are not significantly different; however, they have different pharmacokinetic profiles21. Notably, F(ab′)2 has certain advantages over the parent IgG molecule, for example, easier access to extravascular tissue and rapid elimination from the blood21, higher distribution rate and neutralization potency for low molecular mass venom toxins22 particularly against the venoms of Elapidae family of snakes comprised of relatively higher proportion of low molecular mass toxins25.The antivenoms are manufactured as undigested immunoglobulin G (IgG) molecules14. Because experimental error or insufficient quality control during the preparation might lead to incomplete or partial digestion of IgG proteins or contamination of F(ab′)2 with the undesirable non-IgG proteins of serum, the SL PAV was analyzed by LC–MS/MS to determine the presence of any contaminating serum proteins19. According to WHO guidelines, total protein concentration of PAV solution should not exceed 10 g/dL14. The protein concentration of the aqueous solution of B1 and B2 of SL PAV was found to be 7.3 ± 0.2 and 7.5 ± 0.4 g/dL, respectively indicating they accomplish the WHO guidelines14. The LC–MS/MS analysis of the SL PAV batches against Equus caballus (taxid 9796) proteins in the UniProt database by using emPAI-based label-free quantification method showed that the percent content of immunoglobulin molecule [IgG and/or F(ab′)2] in B1 and B2 of SL PAV was 88.8% and 97.2%, respectively, and the remaining contaminating proteins were fibronectin, fibrinogen, haptoglobin, serum albumin, alpha-2 macroglobulin, albumin, prothrombin, and plasminogen 2 is required. As per the recommendation of WHO (2016)14, the albumin concentration should not be more than 1% of total protein content of antivenom. In the present study, the albumin content of B2 is found within this limit however, this content is slightly higher in B1  followed by SDS-PAGE analysis of the gel filtration protein peaks. The gel filtration chromatogram of each batch of SL PAV was resolved into a major broad protein peak (GF1) that eluted around fractions 42–68 mL 2 and/or IgG 2. In reduced condition, affinity-purified horse F(ab′)2 resolved with a single band ~ 25 kDa as both the heavy and light chains are of same molecular mass (25 kDa); however, the tested PAVs showed a major broad band at ∼ 25 kDa along with a light-stained band at ∼ 35 kDa suggesting some of the F(ab′)2 molecules were incompletely digested in the PAV preparation    content28. The protein aggregates are responsible for early adverse reactions14, though customization and refinements in the antivenom production protocol can lead to pure, aggregate-free F(ab′)2 antivenom31.The densitometry analysis of the SDS-PAGE protein bands of both batches of SL PAV and their respective GF peak (GF1) suggested that they contained minor amounts of undigested IgG 10.0–12.2%) along with F(ab′).2% along2, the latter product shows less complement activation32. While partially digested IgG was present in the PAV in this study, further precision is recommended in this step to improve the product quality.The presence of incomplete or partially digested IgG in SL PAV was determined by enzyme-linked immunosorbent assay (ELISA) and western blot analyses using anti-Fc antibody. The ELISA and immunoblot analysis of the two batches of SL PAV showed 13.9 ± 0.6% and 12.1 ± 0.5%  of undigested IgG content  induced by complement activation34. Studies have shown that, both IgG and F(ab′)2 antivenoms showed higher complement activation when precipitated with ammonium sulphate salt compared to precipitation by caprylic acid35. Nevertheless, IgG/F(ab′)2 aggregates, an excess amount of preservative (cresol), and the hinge portion of the IgG can also contribute to complement activation in PAV-treated patients37. The two batches of SL PAV showed complement activation in vitro with a CH50/mL value (%) of 61.6 ± 3.2, and 64.5 ± 2.9 and AP50/mL value (%) 69.1 ± 4.1 and 65.7 ± 3.9 for SL PAV B1 and B2, respectively  of Indian PAVs in the range of 45 to 70% and 30 to 60%, respectively28. Both the IgG and F(ab′)2 molecules can activate the complement pathway, though the former compared to later showed higher complement activation in vitro32. Noteworthy to mention, the WHO has not fixed any standard safe range for complement activation by antivenoms15. The complement activation shown by SL PAV in this study was assessed only by in vitro analysis and this should be confirmed and validated by in vivo pre-clinical studies or clinical case studies from antivenom-treated patients.The complement activation property of antivenom is primarily due to the Fc portion of the undigested IgG38. Although no adverse effect of IgA has been reported, IgE contamination in antivenom preparations can induce a hypersensitivity reaction leading to anaphylactic shock in antivenom-treated patients39. The two batches of SL PAV were found to contain IgA  and above this level, a high risk exists for inducing pyrogenic reaction in antivenom-treated patients41. The endotoxin load in the two batches of SL PAVs was determined to be within the range of 1.5–1.7 EU/mL, suggesting that they are safe (in terms of endotoxin contamination) to administer to snakebite patients The number of vials of PAV required to cure a snakebite patient depends on the severity of the envenomation (amount of venom injected into the victim), efficacy of the antivenom, and sometimes repeated doses of PAV are administered over a period of 2–3 days. Therefore, to determine any possible deleterious effects of PAV, mammalian cells were challenged with a high dose (20 µg/mL or 2 mg/dL) of SL PAV and cytotoxicity, if present, was assessed up to 72 h post treatment. None of the tested mammalian cells showed cytotoxicity . The in vitro neutralization of selected enzymatic activities and pharmacological properties of venom samples can also be evaluated. The results of these tests can provide a clear understanding of the efficacy of antivenom. These assessments are highly recommended as they involve a minimal use of experimental animals and they are ethically acceptable53.For treating snakebite in SL, Indian PAV is mostly used, though its efficacy and neutralization potency towards SL snake venoms is of immense concernE. carinatus and B. caeruleus, respectively  demonstrated significantly higher immuno-recognition of the SL snake venom samples; PAVs demonstrated the highest and lowest immuno-recognition against the venom of ely Fig. . Furtherely Fig. . Though,55. The results of immunoblot analysis were also in accordance with the ELISA results  exhibited significantly higher immuno-recognition of low molecular mass proteins of SL snake venoms. Moreover, no batch to batch variations (among B1 and B2) were seen in the immuno-recognition properties of country-specific SL PAV , snake venom metalloprotease (SVMP), and snake venom serine protease (SVSP), which play an important role in the toxicity of venom63. Therefore, assessing the in vitro neutralization of catalytic activities and the pharmacological properties of these enzymes by commercial PAV is another important in vitro method for determining the efficacy of commercial antivenom.Snake venom enzymes, particularly those from the Viperidae family of snakes, play a vital role in venom-induced toxicity and lethalityl-amino acid oxidase, hyaluronidase, and SVMP) were better neutralized in comparison to low molecular mass venom toxins , though the neutralization potency of both batches of SL PAV was higher (p < 0.05) than that of Indian PAVs  for PLA2 is immuno-recognized by the SL PAV; however, the catalytic site of PLA2 enzyme, may be due to its poor immunogenicity, is not well neutralized44. The enzymatic activity exhibited by HHV was better neutralized by SL PAV than by Indian PAVs since the latter do not contain antibodies against this venom. This result shows a significant improvement in the immuno-recognition and in the neutralization of venom enzymes of SL snake venoms by country-specific SL PAV in comparison to Indian PAV developed against the Indian ‘big four’ snake venoms. In a nutshell, the in vitro laboratory tests provide convincing evidence for the significant improvement of the venom recognition property of the newly developed SL PAV, in comparison to Indian PAV.The in vitro neutralization of some enzymatic activities and pharmacological properties of SL snake venoms by country-specific SL PAV, and Indian PAV was compared. The enzymatic activities exhibited by high molecular mass venom proteins  and a lower neutralization efficacy against NNV, compared to PSVPL SL PAV.Assessing the neutralization of in vivo lethality and toxicity of snake venoms by commercial PAV is the gold standard for determining their efficacy and it is a crucial step in the clinical assessment of PAV71. In another study, the average venom yields have been reported as 126.32 ± 4.31 mg, 8.352 ± 0.64 mg and 75.98 ± 3.66 mg for NNV, BCV and DRV, respectively72. Thus, these two reports indicate a wide variation in the average venom yield for the same species of snakes in India probably due to difference in regions of study and/or age and size of the snakes. Further, the amount of venom that can be injected by envenomation into a patient would be expected to be proportional to the venom content (yield) of the species of snake. Therefore, the neutralization potency of PAV against DRV, HHV, and NNV should be higher than that against ECV and BCV, because the venom yield per bite of these two snakes should therefore be lower than the former three species of snakes77. However, after the product (SL PAV) registration in SL further clinical trials may be undertaken for the effective clinical dose adjustment, if necessary.For the treatment against snakebite to be effective and convenient to the clinicians, the neutralization potency of PAV should preferably be adjusted according to the venom yield of snakes. It is further to be mentioned that the neutralization potency of the newly developed SL-specific PAV was based on the average venom yield of prevalent snake species with an intention to assist clinicians of SL who are well versed with the use and dose-regimen of Indian PAV. Hospital management of snakebite with similar dosage of SL-specific PAV compared to Indian PAV (which has been used for snakebite treatment by clinicians in SL for decades and they are well versed with the dosages) would be expected to be easier and more clinically relevant than the currently used Indian PAV. The published report on average venom yields of Sri Lankan species of snakes is not available, however, the same species of snakes in India have reported average venom yields of 330 mg, 22.7 mg, 200 mg and 6.25 mg for NNV, BCV, DRV and ECV, respectivelyDendroaspis sp.), cobras (Naja sp.), and kraits (Bungarus sp.) induces a neurotoxic effect that is the primary cause of lethality; albeit, elapids also contain cytotoxins in their venom which lead to significant tissue necrosis79. In this case, the neutralization of lethality would be an ideal model for assessing the pre-clinical efficacy of antivenoms. Nevertheless, the Viperidae snakes show a wide range of pathophysiological effects such as myonecrosis, dermonecrosis, hemorrhage, edema, coagulopathies, bleeding in various organs, hemodynamic disturbances, and renal disturbances81. These effects are cumulatively responsible for the venom-induced lethality. Therefore, in the case of Viperidae venoms, pre-clinical evaluation of antivenoms by assessing the neutralization of lethality is not sufficient for an integrated evaluation of the antivenoms66. The WHO has recommended assessing the neutralization of other toxic activities with supplementary tests and the essential test of neutralization of lethality prior to marketing new antivenoms or introducing existing antivenoms into new geographical locales14.Envenomation by the Elapidae family of snakes, such as the mambas  of SL PAV; however, for an affirmative conclusion regarding batch-to-batch variation, future studies with more number of PAV batches are warranted. However, following the good manufacturing practice (GMP) by the antivenoms manufacturers can result in production of different batches of PAV with uniform potency and compositionNaja naja (NNV), (ii) Daboia russelii (DRV), (iii) Bungarus caeruleus (BCV), (iv) Echis carinatus (ECV), and (v) Hypnale hypnale (HHV). The venoms were collected from snakes of either sex of different age from various locations around Kandy city in SL as per prevalence of snakes in that area. The Saw scaled vipers (ECV) were collected from North SL (Jaffna). The collected venoms were stored lyophilized, and sent by University of Peradeniya (SL) who are collaborating with Premium Serums and Vaccines Pvt. Ltd. The equine PAV developed against SL snakes was obtained from Premium Serums and Vaccines Pvt. Ltd. (PSVPL), India . The PAVs produced against the ‘big four’ venomous snakes of India were obtained from PSVPL, India (Batch no-ASVS (I) LY-014; expiry date: December 2022), VINS Bioproducts Ltd. (VINS), India , Bharat Serums and Vaccines Ltd. (BSVL), India . All of the PAVs used in this study contained pepsin digested and caprylic acid purified IgG [F(ab′)2]. Affinity-purified horse F(ab′)2 and horse IgG were purchased from Jackson Immuno Research Inc, USA and BioRad, USA, respectively. Anti-goat erythrocyte polyclonal antibody (raised in rabbit) was obtained from Fitzgerald Industries International, USA. The Pierce LAL Chromogenic Endotoxin Quantitation Kit was from Thermo Scientific, USA. The NHS-activated Sepharose 4 Fast Flow matrix was purchased from GE Healthcare. All other chemicals were of analytical grade and obtained from Sigma-Aldrich, USA. Human embryonic kidney 293 cells (HEK-293T), murine hepatoma Hepa 1–6 cell line (Hepa1-6), and differentiated rat skeletal (L6) myoblast cell lines were procured from ATCC (American type culture collection), USA.The following five snake venoms (pooled from 5 to 6 snakes) from SL origin were used in this study: (i) 16. PAVs were dissolved in 10 mL of sterilized de-ionized water  and the turbidity of the solution was assessed by a turbidimeter 16. The 2 mL PAV solutions were transferred to pre-weighed microfuge tubes and after centrifugation of the tubes at 10,000 rpm for 10 min, the solutions were decanted, and the tubes were dried in vacuum and weighed again to determine the presence of the insoluble component, if any. The pH of the aqueous PAV solution was determined using a digital pH meter . For determining the residual moisture content, a measured amount of PAV was heated at 105 °C for 3 h in an oven and moisture content was determined by heat drying method82.The texture, colour, and homogeneity of the PAV preparation were determined by visual inspection. The examination of textures like macroscopic collapse, color uniformity, cake shrinkage formation and adhesion properties of powder to the vials of PAVs was done by visual inspection and photographs of vials were also captured83 and the total protein concentration (in g/dL) post dissolving the PAV content in 10 mL of water was determined19. The homogeneity of the preparation was determined by size exclusion chromatography of a 50 mg/mL solution of PAV dissolved in 20 mM Tris–HCl containing 150 mM NaCl, pH 7.4 (equilibration buffer) at 4 °C as described previously84. The size exclusion protein peaks and the crude PAV along with horse IgG and its pepsin digested product F(ab′)2 were separated by 12.5% SDS-PAGE under reduced and non-reduced conditions85. Protein bands were visualized by Coomassie Brilliant Blue R-250 staining. After scanning the gel, intensities of the protein bands were determined by ImageQuant TL 8.1 software 19. Considering that the molecular mass of F(ab′)2 is 100 kDa, the F(ab′)2 aggregate content of the PAVs was also analyzed by densitometry analyses of the SDS-PAGE protein bands of a mass greater than 100 kDa28. The percent of aggregate content (band intensities above 100 kDa) in a particular batch of SL PAV was determined from the cumulative band intensity for that particular batch of PAV.The protein content of PAV was determined as described by Lowry et al.28. Hundred microgram proteins of PAV batches were subjected to reduction (10 mM DTT), alkylation (55 mM iodoacetamide) and then digested with trypsin at an enzyme: substrate ratio of 1:30 for overnight (~ 16 h) at 37 °C45. The trypsin-digested peptides were desalted and concentrated using ZipTip C18 tips following the manufacturer’s protocol.The procedures described in this section is adopted from our previous publicationsThe tryptic fragments of PAV were reconstituted in 0.1% formic acid and separated on a Zorbax 300SB-C18 analytical column , coupled to an Agilent 1200 HPLC, at a flow rate of 300 nL/min. The mobile phase gradient for separation of the peptides was set as: 11% B for 5 min, 11 to 25% B for 20 min, 25 to 53% B for 16 min, 53 to 100% B for 5 min, 100% B for 4 min, and then 11% B for 4 min. Solvent A and B were 0.1% formic acid and 80% acetonitrile (ACN) containing 0.1% formic acid, respectively.45.The peptides eluted from the HPLC column were then fed into a Nanomate Triversa , equipped with an LC coupler and electrospray ionization (ESI) nanospray chip. The LC coupler connects the flow from the HPLC to the ESI chip, where the nano-ESI generated ions were transferred into an LTQ Orbitrap Discovery hybrid mass spectrometer . The ionization voltage was set to 1.7 kV and the raw data were acquired in a data-dependent acquisition (DDA) mode by Xcalibur software . One MS survey scan was followed by 5 MS/MS scans with exclusion duration of 120.0 s during DDA. Survey full-scan MS spectra (from m/z 300–2000 with lock mass set to 445.12 corresponding to polysiloxane) were acquired in Fourier Transform (FT) mode with a resolution of 30,000 . Subsequent fragmentation (MS/MS) was collision-induced dissociation (CID) with normalized collision energy set to 35% in linear ion trap mode. The MS/MS triggering conditions were minimum signal intensity of 10 000; charge state of + 2, + 3; maximum injection time for MS/MS was 500 ms; and isolation width was 2 amuEquus caballus (taxid 9796) protein entries of the UniProt database (version 2019_07) using the Mascot search engine . The following search parameters were used; enzyme: trypsin, maximum missed cleavage sites: 2, precursor ion mass tolerance: 10 ppm, fragment ion tolerance: 0.8 Da, fixed modification: carbamidomethylation on cysteine, variable modifications: oxidation on methionine residues45.The raw LC–MS/MS data were processed to generate peak list in Mascot generic format (*.mgf) using ProteoWizard release version 3.0.331. The peak lists were then searched against the 86. The MassSorter v3.1 software (http://services.cbu.uib.no/software/massSorter/downloadMassSorterNew) was used for in silico generation of the observable peptides of the identified proteins19.The relative abundances of the identified proteins were calculated using Exponentially Modified Protein Abundance Index (emPAI)-based label-free quantification techniqueThe relative abundance of a protein (X) in PAV was calculated using the following equation:28. For the ELISA, 100 ng of PAV protein (in triplicate) or BSA  (negative control) or PBS (reagent control) was coated in the 96 wells of the microtiter plate for overnight incubation (~ 18 h) at 4 °C. After blocking the wells with 5% fat-free skimmed milk, 100 µL of anti-horse IgG Fc specific antibody (1:2000 dilution) or PBS (control) was added and the color was developed by adding TMB /H2O2 as the substrate44. Absorbance was measured at 492 nm in a microplate reader . A standard curve of the graded concentrations of purified horse IgG was prepared and Fc content was determined by ELISA under identical experimental conditions. The Fc content of test samples was compared to the standard curve of horse IgG.The incomplete pepsin digestion of IgG (Fc content of IgG) in the PAV preparation, if any, was determined by ELISA and Western blot analysis using HRP (horseradish peroxidase)-conjugated rabbit anti-horse IgG Fc specific antibody 2O2 substrate kit44. The image was photographed, scanned  and the densitometry analysis of the developed blot was processed with ImageQuant TL 8.1 software . The percent content of Fc in the purified horse IgG was considered as 100% and the other values were compared to that28.For the immunoblot analysis, 20 µg of PAV protein (in triplicate) and horse IgG after separation by 12.5% SDS-PAGE (reduced) was transferred to a PVDF membrane in a semi-dry blotter . After blocking the protein unbound portion of the membrane (non-specific binding sites) by 5% fat-free skimmed milk, it was thoroughly washed with PBS-T in an incubator shaker and the membrane was incubated with HRP-conjugated anti-horse rabbit IgG Fc specific antibody (1:2000 dilution) for 1 h at room temperature. After washing, the membrane blot was developed using the TMB/H37. Human blood samples from healthy donor were collected without anticoagulant and allowed to clot at room temperature for 4 h. The collection of blood from healthy volunteers (who were not under medication) was approved by the Tezpur University Ethical Committee and informed consent was obtained from all participants (TU/TUEC/59/08/4017). The serum was separated by centrifugation of blood at 1000×g for 10 min and the normal human serum (NHS) was stored at − 80 °C until further use. Goat blood obtained from slaughter house was collected in 3.8% tri-sodium citrate (9:1 ratio) and the blood was centrifuged at 4300 rpm for 10 min at 4 °C to pellet down the erythrocytes43.Classical and alternative pathways of complement activation were determined by percentage of hemolysis induced by SL PAV (batch 1 and batch 2) samples2+—10 mM Na-barbitone, 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.4).For determination of complement activation by classical pathway (CP) goat erythrocytes were washed with 1× PBS, pH 7.4 for three times, re-suspended in the same buffer and then incubation with anti-goat erythrocyte antibodies (1:500 dilutions) at 37 °C for 15 min. The antibody-sensitized goat erythrocytes were washed with 1× PBS pH 7.4 buffer and 2% (v/v) erythrocytes suspension was prepared in veronal buffered saline  at a final concentration of 2% (v/v). Fifty micro liters of antibody sensitized goat erythrocytes  or goat erythrocytes  were added to wells of a 96-well microtiter plate containing 50 µL of human serum pre-incubated with 50 µl of SL PAV (1 mg) at 37 °C. The mixture  was incubated for 30 min at 37 °C and then 150 µL of VBS2+ was added to each well. The plate was centrifuged at 1300 rpm at 4 °C for 5 min  and 50 µL of supernatant from each well was transferred to a new 96-well plate containing 200 µL of water. The contents were mixed well by shaking, and the absorbance of mixture was measured at 414 nm in a microplate reader. The results were expressed as the percentage of CH50/mL  or AP50/mL  activation compared with NHS incubated with saline (100%).For alternative pathway (AP) of complement activation analysis, goat erythrocytes were washed with 1× PBS, pH 7.4 and re-suspended in AP buffer  or 100 ng (for the ELISA) of BSA was used. The blots were scanned  and their densitometry analysis was done by ImageQuant TL 8.1 software .The occurrence of IgA and IgE in the SL PAVs, if any, was determined by Western blot analysis and ELISA of PAV against HRP-conjugated anti-horse IgA and anti-horse IgE antibodies, respectively (1:2000 dilution) as described previously14. One mg of PAV solution in sterile water was incubated in trypticase soy broth and thioglycolate medium. Control culture flasks were included for each medium. Flasks were incubated at 25 °C or at 35 °C for 14 days in trypticase soy broth and thioglycolate medium to test for fungal and bacterial cultures, respectively, using the appropriate controls. Culture flasks were examined daily for bacterial or fungal growth by checking the optical density in a spectrophotometer at 600 nm.The sterility of the SL PAV was tested according to the WHO guidelines28. Briefly, a standard curve of graded concentrations of E. coli endotoxin was plotted so that the endotoxin concentration in test sample can be determined as low as 0.1 EU/mL. For endotoxin determination, 50 µL of SL PAV samples in triplicate was added to wells of microtiter plate and the plates were incubated at 37 °C for 5 min at dark. Then, 50 µL of Limulus amebocyte lysate (LAL) to each well was added, mixed gently on a plate shaker for 10 s followed by incubation at 37 °C for 10 min. Thereafter 100 µL of chromogenic substrate solution was added to each well and incubated for 6 min at 37 °C. The reaction was stopped by adding 50 µL of 25% acetic acid and the absorbance of mixture was read at 405 nm in a microplate reader . From the standard curve, the concentration of endotoxin in PAV samples was determined. As a negative control, BSA was used.The level of endotoxin contamination in SL PAV, if any, was determined using a commercial diagnostic kit m-cresol) in PAV preparations for their long-term storage was approved by the WHO in 201614. The m-cresol content was determined by reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) of SL PAV on an Acclaim 300 C18 RP-UHPLC column  pre-equilibrated with 60% methanol in 0.1% (v/v) trifluoroacetic acid (TFA) as described previously28. The flow rate was 0.5 mL/min and the column temperature was maintained at 30 °C. The isocratic programme for the mobile phase was optimized for 18 min87. The detection of m-cresol was observed at 254 nm88. The percentage of m-cresol was determined against a standard curve of m-cresol run in UHPLC under identical experimental conditions.The use of a specified amount of preservative (90. The in vitro cell cytotoxicity was determined by incubating 10 μg/mL of PAV or PBS (control) against human embryonic kidney 293 (HEK-293T), murine hepatoma Hepa 1–6 (Hepa1-6), and differentiated rat skeletal (L6) myoblast cells (1 × 105 cells/mL) grown in DMEM for 72 h at 37 °C and 5% CO228. Thereafter, cell viability was determined by the MTT-based method91 and the result was expressed as PAV-induced cell death (in percentage), if any, with respect to control (PBS-treated cells)60. H2O2 was used as a cytotoxic agent (positive control).The in vitro direct and indirect hemolytic activity of SL PAV (20 μg/mL) was determined against washed human erythrocytes45. Briefly, for ELISA 100 ng of venom was coated for overnight at 4 °C in microtiter ELISA plate. After washing the wells by using washing buffer  the wells were blocked by 5% skimmed milk. The SL PAV or Indian PAV (1 mg/mL) were used as primary antibody (1:500 dilutions) and incubated for 2 h with venom or BSA (blank control) at room temperature. For negative control, the venom samples were treated with naïve horse IgG and developed in parallel. After incubation with primary antibody, the excess antibodies were washed using washing buffer and incubated with anti-horse IgG HRP-conjugated secondary antibody (produced in rabbit) for 2 h at room temperature (1:2000 dilutions). The wells are washed properly and TMB/H2O2 substrate was used for development of color. The reaction was stopped immediately by 2 M H2SO4 and the absorbance was read at 492 nm. For presenting the data, the absorbance values of PAV against venom samples was deducted from the absorbance of negative control.The immunological cross-reactivity of SL snake venoms against SL PAV and Indian PAV raised against venoms of the Big Four snakes of India was determined by ELISA and Western blot analysis45. The SDS-PAGE protein bands were transferred to PVDF membrane in a semi-dry gel transfer system  at 1.2 mA/cm2 of membrane area for 2 h. The transfer efficiency was checked by Ponceau S staining of the membranes. The membranes were blocked by 5% skimmed milk  for overnight at 4 °C. Thereafter, the membranes were washed with TBS-T and incubated with primary antibody (15 mg/mL PAVs) at 1:1000 dilutions for 1 h at room temperature. The excess unbound antibodies were washed with TBS-T and incubated with anti-horse IgG ALP-conjugated secondary rabbit anti-horse antibodies for 1 h at room temperature. Thereafter, the blots were developed using 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) kit  and scanned . Venom samples treated with horse naïve IgG served as negative control. Densitometry analysis of the blots was done using ImageQuant TL software 8.1 .Immunoblotting experiments were performed as described previously by resolving the venom proteins in 12.5% SDS-PAGE under reduced conditionsl-amino acid oxidase, fibrino(geno)lytic, metalloproteinase, hyaluronidase, phospholipase A2) and pharmacological properties (pro/anti-coagulant activity) of SL snake venoms by the newly developed SL PAV and Indian PAVs was determined as described previously52. Briefly, venom (10 µg) was pre-incubated with PAV (100 µg) in a predetermined ratio  for 30 min at 37 °C followed by assaying the mixture for enzymatic activities and in vitro pharmacological properties of venom52. The enzymatic activities and pharmacological properties displayed by crude venom were considered as 100% activity and other values were compared to that48.Neutralization of some enzyme activities  guidelines and ethical guidelines of the “Committee for the purpose of control and supervision of experiments in animals (CPCSEA)”  weighing between 18 and 20 g, age 3 to 4 weeks, following the WHO guidelines50 dose of the venom . To determine the LD50, graded concentrations of venom from each species of snake  were injected intravenously into a group of five mice. Animals were observed for 48 h and deaths during this period, if any, were recorded. The LD50 was calculated by the Reed and Muench method (1938) by using the following formula.The stock solution of venom (1 mg/mL) was diluted in normal saline to prepare different concentrations of venom solution (0.8-fold decrement) so that the middle dose was expected to be the LD50) was mixed with graded concentrations (1.5 fold) of the PAV and adjusted to a constant final volume with saline. The mixtures were incubated for 30 min at 37 °C, and then aliquots of a precise volume (maximum 0.5 mL intravenously) of each mixture were injected into groups of 6 Swiss albino mice. A control group was injected with a mixture of the venom “challenge dose” with saline solution alone (no PAV) to confirm that the venom “challenge dose” induces 50% lethality. After injection, deaths were recorded at 48 h (intravenous test) and the results were analyzed using Reed and Muench method. One antivenom ED50 dose is the amount of antivenom, or the venom/PAV ratio, resulting in the survival of 50% of mice injected with a mixture of PAV and a lethal quantity of venom. The ED50 results were expressed as mg of venom neutralized by per mL of PAV and value was calculated using following formula14.A fixed amount of venom  value, a universal unit for comparison of EDTo determine the hemorrhagic activity, five different concentrations of venom solutions (50 μL) or normal saline (control) were injected intradermally in the shaved skin of lightly anesthetized mice (n = 5). After 3 h post injection, mice were euthanized using a carbon dioxide asphyxiation method. The area of the injected skin was removed and the size of the hemorrhagic lesion was measured using calipers in two directions. The mean diameter of the hemorrhagic lesion for each venom dose was calculated and the mean lesion diameter was plotted against each venom dose to determine the minimum hemorrhagic dose (MHD). One unit of MHD is defined as the dose of venom that produces 10 mm diameter of skin hemorrhage.To determine the venom necrotizing activity, the above procedure was followed and the size of the dermonecrotic lesion was measured. The mean diameter of the dermonecrotic lesion for each venom dose was calculated and mean lesion diameter was plotted against venom dose to determine the minimum necrotic dose (MND), which is defined as the venom dose that produces skin necrosis with a diameter of 5 mm.To determine the in vivo defibrinogenating activity, graded concentrations of venom in 0.2 mL of normal saline or only normal saline (control) were intravenously (i.v.) injected in a group of five mice. After 1 h following the injection, blood was withdrawn by cardiac puncture from the anesthetized mice and transferred to glass tubes. Clot formation was observed visually by the tilting of the tubes. The minimum defibrinogenating dose (MDD) is defined as the amount of injected venom that does not show in vitro blood clot formation.94.To determine myotoxic activity, five different doses of venom dissolved in 50 μL of normal saline were injected into the right gastrocnemius muscle in each mouse (n = 5). Control animals received an injection of the same volume of normal saline. Blood was withdrawn from the tail tip of the anesthetized mice 3 h post injection and serum creatine phosphokinase (CPK) activity was determined using a commercial diagnostic kit 95.For the neutralization assay, a challenge dose of venom was incubated with different concentrations of PAV at 37 °C for 30 min and the venom-antivenom mixture was injected into a group of five mice to determine the neutralization of the above mentioned pharmacological activities of venomp value of ≤ 0.05 between two sets of data was considered to be statistically significant.The significance of difference for more than one set of data was analyzed by a one way ANOVA. The significance of difference between two sets of data was determined by Student’s t test, using Sigma Plot 11.0 for Windows (version 10.0). A Authors confirm that all methods were carried out in accordance with relevant guidelines and regulations and informed consent was obtained from all subjects. All the animal experimental protocols were approved by Premium Serum and Vaccines Pvt. Ltd., Pune, India, following the ARRIVE guidelines and guidelines of Committee for the purpose of control and supervision of experiments in animals (CPCSEA). The details regarding ethics committee/PSVPL animal ethical review board approval numbers for animal experiments are mentioned in Supplementary Table Supplementary Information.


This paper investigates the power resource optimization problem for a new cognitive radio framework with a symbiotic backscatter-aided full-duplex secondary link under imperfect interference cancellation and other hardware impairments. The problem is formulated using two approaches, namely, maximization of the sum rate and maximization of the primary link rate, subject to rate constraints on the secondary link, and the solution for each approach is derived. The problem of a half-duplex secondary link is also solved. Simulation results show that the sum rate and exploitation of the full-duplex capability of the secondary link are strongly affected by both the problem objective and hardware impairments. Spectruapproach  due to tMost studies on BC-aided secondary transmission have focused on a half-duplex (HD) secondary link (SL) ,16,17,18Since FD-based, BC-aided, SL transmission has not been considered in existing BC-aided transmission research, the effects and influence of hardware impairments, including imperfect SIC, have not been studied either in the CRF with FD-based, BC-aided, and SL transmission. Moreover, communicating devices have other imperfections within their hardware , which aIn this paper, we solve the power resource optimization problem for the new CRF with a symbiotic, FD-based, BC-aided SL protecting a primary link (PL), considering HIs for the nodes of both links. In the proposed CRF, the SL consists of two FD nodes that simultaneously transmit to each other. On both the PL and SL, information is sent via a direct link and a BC-aided tag link. We consider various HI cases: imperfect SIC (Imp-SIC), imperfect successive interference cancellation (Imp-SuIC), and other HIs causing signal distortion and channel estimation error. The power resource optimization problem is solved using two approaches with different objectives: (a) maximizing the sum rate (MSR) and (b) maximizing the PL rate subject to rate constraints on the SL (MR). The solution for the FD mode is obtained with a simple root-finding algorithm, while that for the HD mode is derived in a closed form. Through simulation, we show that the sum rate and the exploitation of the FD capability of the SL are strongly affected by both the problem objective and HIs.The remainder of the paper is organized as follows. Recent studies related to this work are reviewed and discussed in Notations: X. In this section, the related works focus on BC, unidirectional (one-way) and bidirectional (two-way) FD and HD communication systems. A time-sharing HD CRF between PL transmission and BC-aided SL transmission is presented in . The autIn , the autAn FD, unidirectional BC-aided PL transmission is presented in . The autAs evident by the related works discussed, most papers concentrate on HD and unidirectional BC-(transmission-aided) PL and SL communications ,24,25,26T. The tag BC link helps improve the spectral efficiency of both the PL and SL due to improvement in diversity gain [t represents the tag T). A BC-aided CRF consisting of a PL and an FD-based SL sharing a spectrum band is illustrated in ity gain , we consz  the interference of the SL within the PL. Hence, the PL transmit power is not considered a variable in the optimization problems.).The system sum rate is maximized by optimizing the power resources of the SL, specifically, The solution to the MSR problem is presented in Theorem 1 below, and the proof is given here.Theorem 1.The optimum for the MSR problem is obtained atandwhere Proof. The MSR problem given in  is non-cSetting Now, let We then substitute the expressions for  Problem  and diffRemark 1.The MSR approach does not promote any prioritization with a performance guarantee between the PL and the SL. For example, This problem is expressed as6maximizeTheorem 2.The optimum for the MR problem is obtained at respectively, withwhere Proof. In the MR problem given in , the maxIn the HD operation of the SL, we assume that the STs perform two-way data transmission on a time division basis, while the PL continues transmitting data throughout the whole communication time. The rates for The solution details of the MSR and MR problems for the HD mode of the SL are presented here. The MSR problem can be split into two subproblems: a subproblem for Similar to the MR problem in the FD mode, the SINR thresholds are applied to determine the Remark 2.We can consider multiple HI scenarios by setting the HI coefficient values accordingly. For the perfect cancellation + no HIs (PSNHI) scenario, all HI coefficients are set to zero. For perfect cancellation + HIs, only the Imp-SIC and Imp-SuIC coefficients are set to zero. For imperfect cancellation + HIs (ISHI), all coefficients are set to non-zero values. In this section, simulation results comparing the FD and HD modes of the SL are presented. Two HI scenarios (PSNHI and ISHI) are considered in addition to varying channel estimation errors. The node-to-node  are randomly distributed within a specified radius from the BC tag, as shown in the example in The effects of increasing Next, we investigate the effects of imperfect cancellation (Imp-SIC and Imp-SuIC) and channel estimation error on the sum rate by varying the HI coefficients e of σe2 . Under bA comparison of our proposed scheme (Sch) to the naive (Com) fixed interference power (d β=0.5)  and fixeshown in , which ishown in . Therefoshown in  and loweshown in .We also consider the influence of time and spatial correlation in Rayleigh fading channels on sum-rate, PL and SL rates, and the STs transmit power as presented in ρ . It can Finally, the performance comparison of the proposed CRF between correlated (Corr) and uncorrelated (UnCorr) Rayleigh fading channels is discussed. For time domain correlation, we consider a channel sampling rate of 50 Hz and a Doppler shift (DS) of 5 Hz. For spatial correlation of channels, we set This paper has investigated the power resource optimization problem for a CRF with a symbiotic BC-aided FD-based SL under various HI cases. In the proposed CRF, the SL consists of two FD nodes and both the PL and SL send information via a direct link and a BC-aided tag link. The problem was solved using two approaches: maximizing the sum rate and maximizing the PL rate subject to rate constraints on the SL. The solution for the FD mode was obtained with a simple root-finding algorithm, while that for the HD mode was derived in a closed form. The simulation results show that the sum rate and exploitation of the FD capabilities of the SL depend on both the problem objective and the HIs.Extensions of this work can be the consideration of the maximization of the minimum rate of the system to identify further improvements in system performance. The replacement of the BC technology with intelligent reflective surfaces (IRS), multi-antenna systems, and interference at the secondary network from the PT are also potential extensions of the current work.


Neuroendocrine neoplasms (NENs) are a heterogeneous group of tumors, their treatment being challenging and requiring a multidisciplinary approach. Though the only curative treatment is surgery, up to 50% of patients are diagnosed with metastatic disease. In the last years, neoadjuvant chemo(radio)therapy has become part of the standard of care in the treatment of different cancer types. However, evidence of its efficacy and safety in NEN patients has not yet been confirmed in the literature. The aim of the present review is to perform an extensive review of the scientific evidence for neoadjuvant therapy in patients with gastroenteropancreatic and thoracic NENs. Although neuroendocrine neoplasms (NENs) are considered rare malignancies, their incidence has rapidly increased in the last decades. Since several patients are diagnosed with metastatic disease, curative surgery is usually not an option , palliatPancreatic neuroendocrine neoplasms (pNENs) account for 1 to 2% of all pancreatic tumors and most of them are sporadic and nonfunctioning. Their incidence has been increasing and survival rates, although improved, remain poor compared with other primary sites, with an overall survival of 3.6 years Table 1.Patients who received neoadjuvant or adjuvant therapy had better over-all survival, suggesting that high incidence of micrometasta-sis contributes to the poor surgical outcomes. In addition, the relatively high proportion of margin-positive re-section raises the question of whether there is a role of downstaging with neoadjuvant therapy, aimed at enhancing resection and lowering risk of systemic recurrence.Xie et al. recently described the largest group of pNENs in whom neoadjuvant therapies were used. The study included 4,892 patients who underwent curative-intent surgical resection. Authors showed that neoadjuvant therapy was mainly prescribed in patients <65 years, with grade 3 pNENs localized in the head of the pancreas and associated to the presence of metastasis. In this setting, Authors did not find any significant improvement in OS even in patients with grade 1 and 2 pNENs thus suggesting that neoadjuvant therapy should be used with caution given the lack of conclusive data . It is wPRRT with radiolabeled somatostatin analogues, over the years, has evolved as an important therapeutic option for the treatment of inoperable or metastasized, well/moderately differentiated, NETs, particularly of the GEP Table 1 The administration of chemotherapy prior to surgical resection is increasingly used for patients with adenocarcinomas of the pancreas with the aim to improve surgical results. Similarly, different regimens of chemotherapies have been described as neoadjuvant treatment in pNENs, all these studies being biased by their retrospective design and by the heterogeneity of chemotherapy regimens used Table 1.Few data are available on the effects of association of chemotherapy and radiotherapy as neoadjuvant therapy in pNENs, all these data being mainly obtained from case reports Table 1.Capecitabine combined with temozolomide (CAPTEM) has been frequently used in the treatment of pNENs. In particular, Strosberg et al. demonstrated that CAPTEM regimen was extremely effective for treatment of metastatic pNENs, resulting in an objective response rate of 70% and median PFS of 18 months . These dThe association of PRRT and chemotherapy has also been used as neoadjuvant therapy in pNENs, taking advantage from the radiosensitising effects of 5-FU. In this respect, the combination of PRRT with 177Lu-octreotate and 5FU chemotherapy was found to be effective in five nonfunctioning pancreatic and one duodenum NEN with inoperable disease, resulting in radiological response in all pNENs. Only one patient underwent surgery successfully after treatment and remained 12 months postoperatively alive and free of disease . FinallyAs for pNENs, neoadjuvant treatments have been proposed for other gastrointestinal NENS, data being mainly based on few case reports Table 1.Neoadjuvant chemotherapy or chemoradiotherapy has been anecdotally reported to be effective also in rectal NENs Table 1.The possible role of neoadjuvant PRRT and PRRT + chemotherapy combination was evaluated in small series of advanced gastrointestinal NENs Table 1.Overall, the few reported experiences would suggest that neoadjuvant chemotreatment can be a successful management strategy in esophageal NEN, while too little data are available about chemo/radio-treatment of other non-pancreatic gastrointestinal NENs in a neoadjuvant setting. However, PRRT seems represent an option in selected cases in this context.Lung neuroendocrine neoplasms represent approximately 20–30% of all NENs. Based on clinical, histological and molecular data, lung NENs are classified in two main categories well differentiated neuroendocrine tumors (carcinoids) and poorly differentiated neuroendocrine carcinomas (NECs). Furthermore, lung carcinoids (LC) are classified in typical (low grade) and atypical carcinoids (intermediate grade) and lung NECs in large-cells and small-cells carcinomas . Only low-quality evidence guides the therapeutic management of LC, and everolimus is the only approved drugs. However currently used systemic therapeutic options include somatostatin analogues, alkylating- and oxaliplatin-based chemotherapies and PRRT. Few data are available on the efficacy of neoadjuvant treatment in thoracic NENs and all these data come from small series and case reports Table 2.Srirajaskanthan el al. reported two patients with lung NENs that received a preoperative chemotherapy with 5-FU, cisplatin and streptozotocin, that induced a good response with consequently a curative resection, both patients being disease free at 36 months after surgery . A multiMore data are available on the role of neoadjuvant therapies in LCNEC. In the multicenter retrospective study by Veronesi et al., 15% of 144 patients who underwent surgical resection for LCNEC, received neoadjuvant chemotherapy . In this study no association was found between neoadjuvant chemotherapy and survival except for stage I patients in whom induction or post-operative chemotherapy tended to be associated to a longer OS (OS rate at 3 years 100% vs 58%) . SarkariNENs of the thymus are very rare tumors, accounting for 0.4% of all carcinoid tumors. Based on WHO 2015, also thymic NENs are classified in two main histopathological and clinical categories: well differentiated tumors, typical and atypical carcinoids and poorly differentiated tumors, small cell and large cell carcinoma. These tumors could be associated to ectopic hormonal secretion, in particular adrenocorticotropic hormone secretion or to multiple endocrine neoplasia type 1. The prognosis of patients with thymic NENs is poor because of the high incidence of local recurrence and distant metastasis and 5-year OS vary from 30–70%. As for other lung NENs, few data are available on neoadjuvat therapy in thymic NENs Table 2.Data on neoadjuvant treatment of NEN patients are scanty, mainly based on inhomogeneous and often incomparable retrospective studies of limited numbers of patients and prospective studies are necessary to clarify the role of neoadjuvant therapy in this clinical setting. Available literature on pancreatic NEN patients suggests PRRT to be variably successful as a neoadjuvant approach, as well as chemotherapy to be more promising in patients with advanced synchronous pancreatic NELM, while—in the same setting—combined chemo/radio/PRRT-therapies would not be supported by sufficient evidence. Neoadjuvant PRRT, chemo or chemoradio-therapies have been anectodotally reported to be effective in non-pancreatic gastrointestinal NENs. Among thoracic NENs, neoadjuvant chemotherapy has been inconsistently reported to be beneficial in LCNC patients, also in relationship to disease stage, while little evidence would suggest neoadjuvant treatment to be negligible in thymic tumors. Therefore, it is advisable to use a neoadjuvant approach with caution, as the effects on quality of life and long-term results in terms of prolonged survival remain yet to be confirmed and the choice of this therapeutic approach should be discussed for each single patient in a multidisciplinary setting.AL conceived and authored the final draft of the manuscript. FF, MR, and RM provided further content, added key references, and authored sections of the manuscript. AF and AC revised the manuscript. All authors contributed to the article and approved the submitted version.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.


Activity in numerous brain regions drives heroin seeking, but no circuits that limit heroin seeking have been identified. Furthermore, the neural circuits controlling opioid choice are unknown. In this study, we examined the role of the infralimbic cortex (IL) to nucleus accumbens shell (NAshell) pathway during heroin choice and relapse. This model yielded subpopulations of heroin versus food preferring rats during choice, and choice was unrelated to subsequent relapse rates to heroin versus food cues, suggesting that choice and relapse are distinct behavioral constructs. Supporting this, inactivation of the IL with muscimol produced differential effects on opioid choice versus relapse. A pathway-specific chemogenetic approach revealed, however, that the IL-NAshell pathway acts as a common limiter of opioid choice and relapse. Furthermore, dendritic spines in IL-NAshell neurons encode distinct aspects of heroin versus food reinforcement. Thus, opioid choice and relapse share a common addiction-limiting circuit in the IL-NAshell pathway. The neural circuits underlying rewarding effects of drugs of abuse and natural rewards are not fully understood. Here the authors investigate the role of the infralimbic cortex to nucleus accumbens shell pathway during heroin or food choice in rats. From rodent preclinical models, some components of the opioid relapse circuitry have been established, including projections from the prefrontal cortex to the nucleus accumbens, a proposed final common pathway to relapse. This projection is functionally segregated into a dorsal and a ventral circuit3. Whereas the dorsal component from the prelimbic cortex to nucleus accumbens core (NAcore) drives relapse for multiple drugs of abuse across multiple models4, the function of the ventral projection from infralimbic (IL) cortex to nucleus accumbens shell (NAshell) is less clear. While pharmacological manipulations of the IL and the NAshell suggest a driver function for these individual brain regions in heroin relapse7, a pathway-specific approach for this circuit has not been explicitly utilized in animal models of opioid seeking. Notably, such an approach has revealed a limiter function for the IL→NAshell pathway for both cocaine and alcohol seeking12.Opioid addiction is a major public health threat13. Preclinical studies support this in that rodents typically prefer natural rewards over drug rewards under conditions of mutually exclusive choice–1914. This behavior has been called voluntary abstinence because animals completely refrain from using drugs in the presence of a natural reward option, supporting the use of natural reinforcers in contingency management therapy20. While the neural circuitry controlling relapse after voluntary abstinence is beginning to be mapped22, we know virtually nothing about the neural basis of opioid choice. Importantly, opioid use disorder in humans is characterized by a substantial population of drug users that become dependent on the drug24 and choose opioids over natural rewards. Thus, we wanted to employ a model wherein individual differences in opioid choice would yield a population average of ~50% choice between drug versus natural reward, allowing us to shift choice behavior with functional brain-site manipulations .By comparison to our knowledge of the neural circuitry of relapse, little is known about the neural circuitry underlying the choice between drug versus natural reward. This is striking given that clinical studies have shown nondrug rewards can prolong abstinence from drug use in humans0 , alpha , Pmax (maximum price paid before consumption declines), EV , and choice and relapse behavior indicated that choice and relapse are conceptually distinct constructs. Consistent with this, pharmacological inactivation of the IL cortex increased heroin choice, but decreased heroin relapse, while having no effect on food relapse. However, targeting the IL cortex outputs to the NAshell specifically using a pathway-specific chemogenetics strategy revealed that this pathway is necessary for limiting heroin choice and heroin relapse, but not food relapse. Furthermore, measures of dendritic spine morphology and density in IL→NAshell neurons differentially correlate with specific variables of heroin versus food motivation. The data support a common limiter function for the IL→NAshell pathway in heroin seeking under both choice and relapse conditions.Here we investigated the role of the IL→NAshell pathway in both opioid choice and relapse in a rat self-administration model that identifies a substantial population of rats that prefer/choose heroin over food reward and results in a population average of ~50% choice for heroin over food. In order to understand whether heroin or food demand predicts choice or relapse behavior, we applied principles from the field of behavioral economics. Relationships between Qt-test: t(26) = 6.34, p = 5.12 × 10−7) and normalized nPmax  = 4.60, p = 4.78 × 10−5) and Omax values  = 7.64, p = 2.10 × 10−8). In addition, demand was less elastic for heroin than food as indicated by lower α values  = 11.2, p = 9.70 × 10−12). Moreover, the unit-independent EV which allows for direct comparisons between different reinforcers25 was significantly higher for heroin  = 7.54, p = 2.61 × 10−8). Finally, consumption at low prices (Q0) for both heroin and food indicated that rats respond significantly more for food than for heroin under these conditions  = 4.87, p = 2.40 × 10−5). Altogether, these results indicate that rats have a higher motivation for heroin and that heroin is considered more essential at higher price points than food reward in this model.Representative demand curves for heroin and food self-administration over increasing price points from a single animal are shown in Fig. Following the behavioral economic phase, choice procedures commenced, and rats had to make mutually exclusive choices between heroin versus food. After choice stabilized, a range of choice behavior emerged, with a population average of ~50% heroin choice. This model is ideal to examine the neural basis of choice between heroin and natural reward. To examine the relationship between choice and other behavioral measures, rats were arbitrarily subdivided into Heroin preferers (>60% heroin choice) versus Food preferers (<40% heroin choice) groups Fig. . Followi0, which clustered with food variables). By contrast, heroin choice separated along with the first component from all other variables, suggesting that heroin choice is distinct from demand, intake, and relapse , with the exception that food preferring rats had elevated Q0 compared to heroin preferers .We applied a principal component analysis to assess the relationships between behavioral economic variables, total intake of food or heroin, heroin choice, and relapse. Behavioral economic variables, total intake, and relapse clustered along with the first component and separated by reinforcer type (food versus heroin) along with the second component  choice sessions on test days. IL-MUS significantly shifted choice toward heroin  = 2.39, p = 0.038). Following choice testing and reacquisition of food and heroin self-administration, rats underwent cued relapse tests with both levers present simultaneously, as during training. Relapse rates for heroin cues were significantly higher than those for food cues  = 11.70, p = 0.005). However, once again, there were no differences in relapse rates between heroin versus food preferers , reinforcing the notion that choice and relapse are distinct behavioral constructs. Interestingly, IL-MUS reduced heroin relapse , but not food relapse. Thus, endogenous activity in IL cortex limits opioid choice but drives relapse.A separate group of rats was implanted with chronic indwelling cannulae aimed at IL and infused with the GABA-A receptor agonist muscimol  = 2.23, p = 0.044). Chemogenetic inhibition of the IL→NAshell pathway shifted choice toward heroin  = 3.19, p = 0.018) and increased heroin relapse  = 2.73, p = 0.034), without altering food relapse  to the IL→NAshell pathwayADD Fig.  was veripse Fig. .Fig. 4Int-test: t(8) = 3.51, p = 0.008). However, chemogenetic activation of the IL→NAshell pathway using the excitatory Gq-DREADD did not alter heroin choice . Thus, under some conditions, it may be possible to exploit the limiter function of the IL→NAshell pathway to reduce relapse.The efficacy of J60 to induce neuronal activity in animals expressing the excitatory Gq-DREADD Fig.  was veriice Fig. , nor didice Fig.  or food ice Fig. . These dice Fig. , but notr = 0.68, p = 0.043) and negatively correlated with food (log) α value . Further analysis of the cumulative frequency distribution of inter-spine intervals  revealed a difference between heroin versus food preferers, with food preferers having significantly smaller inter-spine intervals . By contrast, spine morphology correlated with measures of heroin, but not food, motivation. Spine head diameter positively correlated with heroin relapse  and negatively correlated with heroin (log) α value . No differences were observed in the cumulative frequency distribution of spine head diameter between heroin versus food prefers . Correlational and principal components analyses support the notion that heroin choice and relapse are distinct constructs, making it all the more striking that the limiter function of the IL→NAshell pathway extends to both forms of addictive behavior. Nonetheless, other IL outputs may mediate heroin relapse, as global inactivation of the IL cortex did not elicit parallel effects on relapse, as it did on choice.These results identify the first neural circuit that acts as an endogenous limiter of heroin seeking, namely the IL→NAshell pathway. These results are congruent with findings indicating that this pathway is recruited by extinction learning to limit cocaine seeking34. It has further been suggested that choice  may reveal human subpopulations that could benefit from contingency management therapy–3735. Corroborating this, preclinical studies consistently show a negative relationship between α and relapse rates38, consistent with our findings here for heroin. However, principal component and correlational analyses of behavioral economic variables, choice, and relapse clearly identified choice as a distinct behavioral construct. Whereas behavioral economic variables associated with each other, predominantly clustering by reward type (heroin versus food), they also clustered with relapse and total intake (consumption history). Thus, these variables likely reflect a larger behavioral construct, possibly motivation. By contrast, the opioid choice fell within its own principal components space, which we propose represents the construct of cognition. While purely theoretical, regardless of the hypothetical construct assigned to principal component space in these analyses, heroin choice clearly separated from the other behavioral variables, including heroin relapse.Behavioral economics has been applied to the study of human substance abuse and was shown, along with choice-based relapse, to relate to measures of dependence and abuse40. Though IL→NAshell activation was not sufficient to limit opioid choice or relapse during acute withdrawal after choice procedures, it was capable of reducing cued heroin relapse in a standard extinction-reinstatement model. It is possible that IL→NAshell activity is already at a ceiling after choice procedures and/or acute withdrawal, but may be further enhanced to limit relapse after extinction and/or protracted withdrawal. Interestingly, global IL-MUS inactivation actually decreased heroin, but not food, relapse, an effect opposite that of IL→NAshell pathway-specific inhibition. This suggests that the IL cortex can also function as a driver of heroin seeking, consistent with other reports7. Nonetheless, our findings in this heroin model are consistent with observations that IL neuronal ensembles exert inhibitory control over alcohol seeking42. As specific IL outputs were not identified in the latter studies, these apparent discrepancies may relate to different IL outputs being engaged under different conditions.Since global inactivation of IL cortex with IL-MUS produced a similar upward shift in heroin choice as IL→NAshell pathway-specific inhibition, the opioid choice may predominantly recruit this IL output (NAshell). Similarly, IL→NAshell pathway inhibition produced an increase in heroin, but not food, relapse. Thus, the IL→NAshell functions as a common limiter of these distinct behaviors and may reflect the top-down cognitive control IL exerts over NAshell-motivational processes43. Although the IL cortex has not to our knowledge been examined in the context of opioid choice, within the orbitofrontal cortex, which receives input from the IL cortex44, distinct populations of neurons encode food or heroin choice45. The adjacent anterior insular cortex and its projection to the central amygdala regulate methamphetamine relapse following voluntary abstinence by food choice22, and expression of a ɣ-aminobutyric acid (GABA) GABA transporter (GAT-3) in the central amygdala has been linked to alcohol choice in both rodents and humans46. Other anterior insular projections to the NAcore mediate choice between two distinct food rewards, and downstream μ opioid receptors in the NAcore are involved47. Mu opioid receptors are also highly expressed in the NAshell, and concentrated in hedonic hot spots which directly mediate opioid and food reward–5048. Activation of these μ opioid receptors reduces neuronal activity and increases food consumption, an effect that is consistent with other pharmacological means of inhibiting NAshell activity52. This may in part account for observations that prolonged heroin self-administration, which directly activates μ opioid receptors, increases motivation for both heroin and sweet reward29.Surprisingly, very little is known about the neural circuits controlling the choice between opioid versus natural reward. However, choice behavior likely involves distinct prefrontal circuits that attribute and compare the expected value of different rewards, and that monitor behavioral strategies. Within the IL cortex, neuronal ensembles encoding alcohol versus saccharin rewards (two tastant rewards) were found to exhibit some overlap, but an equal portion of these ensembles were distinctAnalyses of dendritic spines suggest that specific structural features in IL→NAshell neurons may encode distinct aspects of heroin versus food reinforcement, with spine head morphology correlating with heroin measures (heroin relapse and demand elasticity), and spine density with food measures (food relapse and food elasticity). Opioid choice, by contrast, may specifically relate to the inter-spine interval, with lower opioid choice reflected in more closely spaced spines along the dendritic shaft. How these structural markers relate to the physiological activity of these neurons, and the specificity of these markers to the IL→NAshell pathway remains to be determined. It is nonetheless striking that these post hoc structural analyses distinguished heroin versus food relapse in morphological versus densitometric domains, despite the fact that heroin relapse correlated with food relapse in this study . Notably, however, heroin and food relapse did not correlate in the IL-MUS study . Thus, presenting each lever independently as in the former study may have resulted in greater generalization across levers, even though the order in which they were presented was counterbalanced. This may also explain why heroin and food relapse rates did not differ in the behavioral economics study, but did in the IL-MUS study. When both levers were presented simultaneously as in the latter study, thus forcing the rats to choose how they allocated their seeking behavior, heroin relapse rates were higher than those for food.53 due to the majority of rats exclusively choosing food reward over drug reward. Still, others have been able to produce these subpopulations by increasing the delay to food reward 54 or by extending the duration of daily drug self-administration –5755. Our model yields ~50% heroin choice  regardless of whether we used a behavioral economics model where animals had an extensive opioid self-administration history versus an abbreviated choice model with a more limited history of opioid exposure. This affords us a unique opportunity to examine neurobiological differences in these subpopulations going forward. Future studies should examine how the IL→NAshell pathway fits within a broader circuitry controlling choice between opioid and natural reward, and further probe the circumstances under which this pathway is capable of limiting opioid addiction-like behaviors.In our choice model, we identified subpopulations of heroin preferring versus food preferring rats under equivalent price conditions (FR3). This contrasts with findings from other choice models, which have been dubbed voluntary abstinence models58. Rats were excluded from the final dataset if catheter patency was lost, if they were identified as outliers during statistical analysis , if they failed to meet specified behavioral criteria, or for poor placement and/or virus expression (where relevant).All animal procedures followed guidelines approved by the University of Colorado Denver, Anschutz Medical Campus Institutional Animal Care and Use Committee. Subjects were age-matched (P55-60) male and female Wistar rats . Animals were single-housed in a temperature and humidity-controlled environment (lights on 7 a.m.–7 p.m.) with free access to standard laboratory chow and water. Our procedures followed the guidelines outlined in the Guide for the Care and Use of Laboratory Animals59 using the following coordinates relative to bregma: NAshell +1.4 mm anterior-posterior (A/P), ±0.8 mm mediolateral (M/L), and −6.0 mm dorsoventral (D/V); IL +2.9 mm A/P, ±0.6 mm M/L, and −2.5 mm D/V. Carprofen (5 mg/kg) and cefazolin (30 mg/kg) were administered to alleviate surgical pain and prevent infection. Rats were allowed to recover from surgery at least 1 week prior to virus infusions.Rats were anesthetized with ketamine/xylazine (80/7 mg/kg) and implanted with an intravenous catheter in the right jugular vein and intracranial cannulas n = 30; Salk Institute; titer 1.1 × 1012 GC/ml). The IL was injected with viral vectors carrying inhibitory or excitatory designer receptors exclusively activated by designer drugs . The DREADD ligand JHU37160 dihydrochloride , dissolved in sterile water, was administered at a dose of 0.1 mg/kg  15 min prior to testing60.Under light isoflurane anesthesia, rats received bilateral injections of the virus into IL and NAshell . Injectors extended 3 mm beyond cannula for IL and 2 mm for NAshell. The NAshell was injected with a retrogradely transported AAV vector carrying Cre recombinase  prior to histological procedures in order to visualize the spread of MUS-TMR-X at the injection site62.Rats received bilateral microinjections of muscimol  or vehicle (sterile PBS) into the IL cortex through their intracranial cannula just prior to testing. Injectors extended 3 mm beyond the cannula. The dose of MUS (15 ng/side) was based on previous work indicating this dose does not produce locomotor effectsFood self-administration training (1 h/session/weekday) occurred in standard operant chambers inside ventilated, sound-attenuating cubicles . Rats were trained to self-administer food pellets  on a fixed-ratio 1 (FR1) schedule of reinforcement. During food self-administration (1 h/session/weekday), only the right retractable lever was extended. Three pellets and a 3.5 kHz tone cue (5 sec) were delivered each time the FR requirement was reached, and the lever retracted during cue presentation. After five sessions, training progressed through five additional FR schedules  with at least 2 days on each FR, or until ≤20% variability in rewards earned was achieved.After completing food self-administration, rats began daily heroin self-administration training , during which only the left lever was extended. Heroin  was dissolved in 0.9% sterile saline. After an FR requirement was reached, a heroin infusion (0.04 mg/50 µl/2.85 sec) was delivered, a light above the left lever was turned on (5 sec), and the lever retracted during cue presentation. After at least five sessions, animals progressed over additional FR schedules , with at least 2 days on each FR, or until ≤20% variability in rewards earned was achieved. To prevent infection and catheter occlusion, cefazolin and taurolidine-citrate solution were administered after each self-administration session.28. Food and heroin demand curves were generated separately for each animal in Matlab , using the average responding over the last two sessions on each FR, according to the formula63:We used a between-session behavioral economics paradigm where male rats were allowed to self-administer food or heroin in daily sessions of fixed duration38:) and constant k (set to 8.85) specifies the range of consumption values. From the resulting demand curves, we extracted the following variables: α , Q0 , Pmax , and Omax (the maximum rate of responding to defend desired consumption)64. We also calculated EV according to the formula25:C indicates consumption  or heroin (0.04 mg infusion + heroin-light). During each trial, both right and left levers were extended, and the rat needed to reach an FR3 requirement on one of the levers to earn a food or heroin reward. Afterward, both levers retracted for a 10 min time-out between trials (house-light off and levers retracted). The first five-choice sessions contained two forced-choice trials for each reinforcer  during which only one lever was extended and ten free-choice trials with two levers. Forced choice trials were administered in the order: food-heroin-food-heroin. Afterward, rats moved to free-choice sessions  for the remainder of choice training. Following at least three free-choice sessions or until there was ≤20% variability in total choices over the last two sessions, rats started choice tests, separated by at least two stable choice sessions. Prior to each test, rats were injected with J60 or vehicle in a randomized, counterbalanced order. The choice was expressed as the ratio of heroin choices over total choices.For the IL-MUS experiment, we used a protocol wherein male and female rats were trained to self-administer food and heroin simultaneously during daily self-administration sessions. The parameters were equivalent to those described above for the behavioral economics experiment, except that sessions were a total of 3 h long, and the number of self-administration days was held constant across all rats. After six sessions on an FR1 schedule of reinforcement, rats progressed to an FR3 scheduled for three sessions and then proceeded to free-choice sessions thereafter. These free-choice sessions were identical to those used after behavioral economics procedures except the session duration was held constant at 3 h . After at least nine sessions on choice, and meeting stability criteria , rats began testing after intra-IL microinfusion of the vehicle or MUS . Rats underwent two choice sessions in between test days using a within-subject design with treatment order counterbalanced across subjects.After rats completed choice testing, they were returned to self-administration procedures to ensure recent exposure to both heroin and food (and their respective cues) across all rats (including heroin and food preferers). After three FR3 reacquisition sessions, rats began cued relapse testing. Thus, the relapse terminology used throughout this manuscript refers specifically to relapse during acute withdrawal . For the behavioral economics study, each lever was extended separately in two 30-min bins, separated by a 10 min time-out . Pressing on each lever resulted in cue delivery (food-tone or heroin-light) on an FR3 schedule. Levers retracted during cue delivery, as during self-administration sessions. The order in which the food or heroin lever was presented was counterbalanced across rats and tests. For the IL-MUS experiment, the relapse test was conducted with both levers (food and heroin) available simultaneously for the 1-h test session. Each rat received drug (J60 or IL-MUS) or vehicle treatments in a randomized, counterbalanced order separated by at least two FR3 reacquisition sessions.Rats were transcardially perfused with 0.9% saline followed by 10% formalin. Brains were extracted, post-fixed in formalin, cryoprotected in 20% sucrose, and sectioned (40 µm) on a cryostat . Free-floating sections were blocked in 2% normal donkey serum (NDS) in phosphate-buffered saline with 0.3% Triton X-100 (PBS-T) and incubated overnight at 4 °C with rabbit anti-cFos  and/or chicken anti-mCherry . Sections were then rinsed in PBS-T and incubated for 2 h in PBS-T with 2% of NDS containing fluorescent Alexa-conjugated secondary antibodies . NeuroTrace fluorescent Nissl 640/660  was applied during the secondary antibody step, or DAPI coverslipping media was used as a counterstain. Images were acquired with an Olympus slide scanner microscope . Ten rats were excluded from chemogenetics experiments based on low or misplaced virus expression.The chemogenetic approach was validated using Fos as a measure of neuronal activity after J60 or vehicle administration in male and female rats. The Gi (hM4Di)- or Gq (hM3Dq)- DREADD was restricted to the IL-NAshell pathway using the combinatorial viral strategy described above (see Chemogenetics approach). To verify Gi-DREADD functionality, rats received an injection of cocaine  to induce Fos, 15 min after J60  or sterile water pretreatment. To verify Gq-DREADD functionality, rats received J60  or vehicle (sterile water). All injections were given in the home cage, and all rats were perfused transcardially 2 h after J60 or vehicle. Histology and immunohistochemistry were performed as described above, and images were analyzed using Imaris software (Bitplane). A region of interest (ROI) was set and the number of Fos+, hM4Di−, or hM3Dq-mCherry+ neurons in the IL was automatically quantified using the Imaris spot detection function on thresholded images. The number of co-expressing neurons in each section were determined using the colocalize spot function. For all rats, the number of cells was averaged within each hemisphere across at least three sections.65. Dendritic segments from IL→NAshell neurons selected for spine analysis were visually connected to the soma. Z-series were acquired with a confocal microscope (Olympus FV1200) using a 60x oil immersion objective (1.4 NA), at 1024 × 512 frame size using 4.1x digital zoom and a 0.1 µm step size. mCherry was excited using a 488 nm argon-ion laser line and overall, imaging parameters were held constant during the acquisition process. Apical dendrite segments from layer 5/6 pyramidal neurons were imaged in layer 2/3 (~300 µm from the soma), ~10 µm before branching into the apical tuft. Images were cropped and deconvolved using Huygens (SVI) software. Dendritic segments (mean ± SEM length: 65.6 ± 0.38 µm) were imported into Imaris (Bitplane) software and spines were semiautomatically drawn using the filament module. Spine density and head diameter values were determined from four dendritic segments per animal.Spine analyses were performed in DREADD-infected, mCherry-tagged neurons from the behavioral economics studyt-tests. Cell counts for Fos expression analyses were analyzed using two-tailed unpaired t-tests. Linear relationships between variables were analyzed using Pearson’s Correlation Coefficient. Principal component analysis was conducted using z-transform scaled data in Matlab. Cumulative frequency distributions were compared using two-sample Kolmogorov–Smirnov tests in Matlab. Outliers were identified using ROUT analysis. Significance was defined as alpha <0.05 and data were graphed as mean ± standard error of the mean (SEM).All statistical tests were performed in Prism  and Matlab . Choice and relapse tests were analyzed using repeated measures analysis of variance (ANOVA), followed by Bonferroni corrected planned comparisons, or by using two-tailed, planned comparison paired Further information on research design is available in the Supplementary InformationReporting Summary