Accidental insulin-induced hypoglycemia in nondiabetic dogs

Accidental insulin-induced hypoglycemia in nondiabetic dogs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Accidental insulin-induced hypoglycemia in nondiabetic dogs Luis Antônio Tondo, Gabriela Oliveira Pereira, Reina Isabel Cartagena, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6613595/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Veterinary Research Communications → Version 1 posted 4 You are reading this latest preprint version Abstract Purpose: Herein, animal cases of insulin-induced hypoglycemia are presented in nondiabetic patients. We report an outbreak of iatrogenic insulin poisoning in dogs. Methods: Twenty dogs presented peracute clinical signs, and seven were submitted for postmortem and histological examination in the Setor de Anatomia Patológica (SAP) at the Federal Rural University of Rio de Janeiro, Brazil. Blood serum samples from the cardiac clot of the right ventricle of the dogs were sent for radioimmunoassay examination for insulin dosage. Vitreous humor samples were submitted for flame atomic emission photometry for potassium level determination. Results: All dogs had previously attended an anti-rabies vaccination campaign on the same day and time and showed peracute clinical signs after the application. The respondents reported suspicions about accidental parenteral administration of insulin in the dogs. Tremors, inability to stand still, tachypnea, and intense vocalization were evident in 2 to 13 hours of clinical evolution. Histologically, there was neuronal necrosis in the temporal and frontal cortex. Radioimmunoassay identified insulin in three of the submitted samples. Flame atomic emission photometry revealed high levels of potassium in all samples. The epidemiological, clinical, and histopathological findings associated with identifying insulin in the blood serum and atomic emission spectroscopy for potassium recovery endorsed the suspicion of insulin poisoning. Conclusion: For the first time in veterinary medicine, accidental insulin-induced hypoglycemia is reported in nondiabetic dogs. Flame atomic emission photometry was a promising tool for indicating high circulating insulin levels in forensic veterinary medicine. Veterinary pathology neuropathology emergency care insulin poisoning humor vitreous potassium Figures Figure 1 Figure 2 BACKGROUND Insulin overdose is a health emergency in dogs and cats. Insulin overdose is more common in cats than in dogs, especially in obese diabetic cats (Whitley et al. 1997 ). Insulin is a hormone with great relevance in glucose metabolism. Exogenous insulin therapy is crucial for managing various diseases in small animals. Otherwise, overuse in a patient can lead to severe problems. In the medical literature, cases of insulin poisoning are scarce and primarily associated with accidental misapplication, criminal acts (Bottinelli et al. 2020 ), or suicidal purposes (Wunder et al. 2014 ). Even to date, murderers related to insulin application are hard to diagnose. The clinical signs are nonspecific, with few postmortem and histopathological lesions, and determining the insulin dosage in blood, urine, vitreous humor, or tissue is challenging (Bottinelli et al. 2020 ). Insulin is an essential hormone that stimulates the uptake and storage of carbohydrates, fatty acids and amino acids (Skowronek 2019 ). The sodium-potassium-ATPase pump is an ATP-dependent cellular mechanism that regulates intracellular osmotic concentrations by mediating sodium secretion and potassium influx. Insulin stimulates the cellular uptake of glucose, triggering the activation of sodium-potassium-ATPase pumps, which leads to the influx of extracellular potassium into the cell. Therefore, extracellular potassium and sodium levels could be potential postmortem biochemical markers for exogenous insulin poisoning, depending on the type of sample selected (Arnolds et al., 2010 ). The application of biochemical analysis in postmortem samples in veterinary medicine is scarce; however, it is broadly used in human forensic medicine. Autolysis and putrefaction directly influence the testing results of postmortem biochemical techniques. Depending on the condition of the samples, the results can be unreliable, making the analysis difficult to interpret (Madea and Musshoff 2007 ; Byard and Tsokos 2013 ; Lutz et al. 1997 ). Vitreous humor is a gel-like substance from the posterior chamber of the eye. It is composed primarily of water, collagen, hyaluronic acid, and electrolytes and is kept in a privileged environment compared to other body fluids. Drug penetration into the vitreous humor through the blood-ocular barrier is possible but depends on the drug diffusion rate (Thevis et al. 2012 ). Consequently, due to its enclosure within the ocular globe, the vitreous humor maintains low interaction with the environment and is relatively stable and resistant to postmortem alterations. This feature makes the vitreous humor a promising candidate for toxicological testing (Chavhan et al. 2014 ). Insulin-induced hypoglycemia is rare in small animal practices and is mainly associated with owners’ misdosage of insulin in diabetic patients (Idowu and Heading 2018 ). Herein, we report an outbreak of accidental insulin-induced hypoglycemia in nondiabetic dogs and the utilization of ancillary testing, which could indicate high levels of circulating insulin in animals. CASE PRESENTATION Ethics. We adopted the CARE Guidelines: Consensus-based clinical case reporting guideline development for transparency of the present case series report. All dog owners authorized the clinicopathological study. Emergency care and therapeutics. The health department secretary of Magé, RJ, reported that 20 dogs that attended the same vaccination post showed similar clinical signs.After the vaccination, the dogs were brought to their homes and showed clinical signs of vocalization, shivering, and obtundation. Three dogs had seizures, and one dog vomited. The clinical course of the neurologic signs varied from 2 to 13 hours. One dog was taken to a veterinary clinic for treatment but died immediately after arrival. After the first clinical signs were detected, the dog owners searched for assistance, and the vaccination campaign was blocked. As the health department secretary informed, there was suspicion of accidental parenteral insulin administration in the dogs due to empty insulin vials among the discarded vaccine vials. It was reported that twenty dogs presented with peracute clinical signs characterized by intermittent seizures, tremors, inability to stand still, tachypnea, and intense vocalization. In total, 15 dogs that attended the vaccination campaign on the morning of September 4 th were submitted for clinical service after presenting post-vaccination neurological signs. One of the authors, Janice Biazzi Pires, reported the clinical approach. All dogs were assisted in a private veterinary clinic's emergency and critical care service on the morning of September 4 th , 2021. The first dog received medical assistance at 9:30 a.m. on September 4 th . Due to the clinical history of prior vaccination, an anti-anaphylactic shock protocol was initiated. The dog was treated with parenteral adrenaline, venous fluid, and diuretics; however, with an unsuccessful outcome. A similar protocol was applied to 8 dogs, and none survived. In 7 dogs, the blood glucose was assessed and revealed values varying from 19 to 60 mg/dl, which raised concern for hypoglycemia. The clinical protocol was reviewed, and intravenous administration of dextrose associated with sedation was initiated. All dogs submitted to the reviewed clinical approach presented a better outcome and survived. Pathology and toxicological sampling. The health department secretary of Magé, RJ, was responsible for sending a picture of two empty regular-acting human insulin vials to the veterinary pathologists as an attachment to the clinical history. Allegedly, 1 mL of regular insulin was administered to twenty dogs due to the dosage recommendation for rabies vaccination. The carcasses of the seven deceased dogs were collected by the team of the health secretary of Magé; however, the dogs' bodies were frozen about 30 minutes after death at the clinic where the emergency care occurred. The dogs' carcasses were kept frozen five days before the necropsy procedure. Seven dog carcasses from different owners were received for necropsy in the Setor de Anatomia Patológica from the Universidade Federal Rural do Rio de Janeiro (SAP/UFRuralRJ). The respective owners informed the history of their dogs. All owners reported that the dogs were sent to an anti-rabies vaccination campaign in Magé, RJ, Brazil. The seven dogs were submitted to the SAP/UFRuralRJ by the health department secretary of Magé, RJ. They were submitted to a detailed postmortem examination where material for forensic toxicology was collected for further analysis. All seven dogs were submitted to trichotomy for investigation of injection sites in the skin. A full set of tissues from all seven dogs (brain, spinal cord, lymph nodes, thyroid, adrenal glands, ovaries/testis, as well as samples of omentum, mesentery, gall bladder, uterus, lungs, trachea, esophagus, stomach, intestines, liver, spleen, kidneys, and heart) was collected in 10% buffered formalin solution and processed routinely for histology. For forensic toxicology testing, right ventricle blood clots and vitreous humor were collected from all dogs. The right ventricle clot was submitted for insulin dosage by radioimmunoassay, and the vitreous humor was submitted for a potassium dosage by atomic flame emission photometry to analyze potassium. To rule out other possible causes of diseases with neurological clinical signs, fresh frontal and parietal cortex, cerebellum, and spinal cord samples were analyzed by qPCR for canine distemper virus (CDV) (Fischer et al. 2013), and formalin-fixed samples by immunohistochemistry for rabies (Pedroso et al. 2008). Out of the twenty dogs, thirteen died, and five of them did not receive medical aid. Seven dogs were submitted for postmortem examination. The seven dogs varied from 1 to 14 years old and were two females (one pregnant) and five males. This group had four mixed-breed dogs: one Labrador Retriever, a Basset-hound, and a Poodle. The weight of all animals varied from 5.1 to 24.5kg. None of the dogs had blood glucose values available during the necropsy. At necropsy, the body condition of the seven dogs ranged from 2.5 to 4 (1 to 5 scale). All seven dogs presented a red dot (local hyperemia) on the skin of the left flank, indicating the putative application site (Fig. 1a). During the vaccination campaign, two empty regular-acting human insulin vials and boxes (Fig. 1b) were found mixed with other discarded anti-rabies vaccine vials. At necropsy, in addition to skin hemorrhage at the site of injection, heterogenic and nonspecific gross lesions were identified in all seven dogs. A grayish irregular vegetative nodule in one dog was attached to the aortic valve (aortic valve endocarditis). Another dog had a red and soft splenic nodule with a 2,5 x 2cm diameter. Also, in this dog, the kidneys were firm and slightly irregular. The pregnant bitch had five well-developed fetuses in the uterus. No other relevant gross lesions were found in the remaining dogs. At histopathological examination, four dogs had significant amounts of amorphous, homogeneous, and eosinophilic material mixed with foamy macrophages within the pulmonary alveoli (edema). The grossly observed vegetative nodule in the aortic valve was characterized by severe neutrophilic and histiocytic inflammatory infiltrates with moderate basophilic cocci-bacilli colonies (bacterial endocarditis). The splenic nodule corresponded to a fairly demarcated neoplasm composed of pleomorphic endothelial cells (hemangiosarcoma). Two dogs had mild alterations in their kidneys. One had a moderate number of lymphocytes and plasma cells surrounding glomeruli. These glomeruli were hypercellular and had mild basal membrane thickening. The other dog had a moderate number of lymphocytes, plasma cells, and histiocytes expanding the renal interstitium. Three dogs in the frontal and temporal cortex presented a moderate amount of retracted, hypereosinophilic neurons with a hyperchromatic nucleus (neuronal necrosis) (Fig. 2ab). Insulin enzyme radioimmunoassay. Right ventricle blood clots from all dogs were collected, kept frozen, and referred to an external laboratory for serum insulin quantification through enzyme radioimmunoassay (Shen et al. 2019). The toxicology examination of the right ventricle clot showed insulin doses in three samples. The values were 27.82 uUI/ml, 11.13 uUI/ml, and 0.24 uUI/ml (Table 1). Analytical procedure for determination of potassium in vitreous humor. Vitreous humor samples from all dogs were collected and referred to forensic toxicology testing. Initially, a calibration curve was made to use the flame photometer (MicroNal brand, model B462). For this purpose, the equipment was zeroed with distilled water, and the suction pressure was adjusted to 0.8 atmospheres. The mother solution was prepared by weighing 50.05g (Gehaka scale BG1000) of potassium chloride and diluted in a 1000 mL volumetric flask, reaching a concentration of 0.05 g/L or 67000 mEq/L. Through this mother solution, dilutions were performed, resulting in six solutions with different concentrations, namely: flask 1 (0.104 mEq/L), flask 2 (1.045 mEq/L), flask 3 (2.094 mEq/L), flask 4 (4.187 mEq/L), flask 5 (8.375), flask 6 (16.755 mEq/L). After building the curve, the analyses were performed. The equipment was reset with distilled water between each analysis to clean it. Reading of analyzed substances: The substances were read from vitreous humor samples of dogs with suspected insulin poisoning and dogs due to natural death with up to 30-minute postmortem intervals. Vials from two dogs (37546 and 37549) had insufficient sample volume for testing. Experimental results: The calibration curve presented a coefficient of determination r2 of 0.9941, showing adequate linearity for the analysis. The chosen range was from 0.1045 mEq/L to 16.75 mEq/L (Table 2). Quantitative PCR (qPCR) for canine distemper virus (CDV): RNA extraction and cDNA synthesis. Fresh frontal and parietal cortex, cerebellum, and spinal cord samples from the seven dogs were collected and submitted to RNA extraction employing the TRIzol® Plus RNA Purification Kit according to the manufacturer's instructions. The resulting RNA was treated with DNase I (Invitrogen™, ThermoFisher) to eliminate contaminating genomic DNA. In addition, the quality and integrity of the RNA extracted were evaluated by a Nanodrop spectrophotometer (Invitrogen™, ThermoFisher) and electrophoresis in 1% agarose gel. The RNA extracted was quantified by Qubit™ fluorometer (Invitrogen™, ThermoFisher) using the Qubit™ RNA broad-range assay kit. The complementary DNA (cDNA) was synthesized with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems™, ThermoFisher) according to the manufacturer's instructions. Subsequently, the cDNA was quantified using a Qubit™ fluorometer (Invitrogen™, ThermoFisher), which employed the Qubit™ ssDNA Assay Kit. A combined reverse transcription polymerase chain reaction (RT-qPCR) method was used for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV), as recommended by Fisher et al. (2013). The assay was performed in a MicroAmp® Optical 96-Well Reaction Plate at StepOne Plus thermocycler (Applied Biosystems®, ThermoFisher). The qPCR reaction was carried out in a final volume of 12 µL, containing 1X TaqManTM Universal Master Mix (Applied Biosystems®, ThermoFisher), 0.4 µM of each primer and 0,20 µM of the probe, and 45 ng/µL of cDNA. Thermocycling conditions were 95 °C for 10 minutes, and 40 cycles of denaturation at 95 ºC for 20 s, annealing/polymerization at 60 ºC for 60 s. Samples with Cq values ≤ 40 cycles were considered positive. All dogs were negative for canine distemper virus in real-time polymerase chain reaction. Immunohistochemistry for rabies. 5µm-thick histological sections of the dogs' brainstem were applied to positive slides. Endogenous peroxidase was blocked by incubating the slides in a 3% hydrogen peroxide solution in distilled water for 15 minutes at room temperature. For antigenic recovery, Citrate buffer was used (2.1g of citric acid in 1 liter of distilled water, adjusting the pH to 6.0 with 0.5% NaOH). The sections were treated with 5% skimmed milk (Molico®) diluted in distilled water for 15 minutes. The sections were covered with a solution containing the primary antibody (GeneTex GTX21002 and Biodesign C86307M) in a 1:1,000 dilution in PBS, incubated in a humid chamber for 12 hours at 4 °C (Pedroso et al. 2008). EnVision secondary polymer (Dako, www.agilent.com) was then applied to the sections in an oven at 37 °C for 30 min. Color development was done with 3,3-diaminobenzidine chromogen (DAB + Substrate Chromogen System; DakoCytomation) for 2 min. A bovine rabies case, previously confirmed by direct immunofluorescence (DIF), was used as a positive control. Finally, the sections were counterstained with Harris hematoxylin and coverslipped. Cases were considered positive where structures morphologically compatible with Negri corpuscles were seen within the neuronal cell bodies. All dogs were negative for rabies. DISCUSSION AND CONCLUSION The dogs presented in this outbreak died due to insulin-induced hypoglycemia, and this highlights the importance of veterinary care during vaccination campaign activities. The clinical and pathological picture was typical of hypoglycemia. Blood glucose measurement is a key diagnostic indicator in cases of suspected insulin poisoning; in the present cases, values ranged from 19 to 60 mg/dL during emergency care, consistent with hypoglycemia and further supported by histopathological findings of neuronal necrosis. The toxicological findings of high levels of postmortem insulin in the right ventricle blood clot confirm high insulin levels circulating. The high potassium values in the humor vitreous confirm the sodium-potassium pump disturbance. Based on multiple diagnosis tools, we confirm that the dogs went into hypoglycemia associated with exogenous insulin poisoning. The confusion between the two vials occurred because the commercial insulin vial was labeled “Insulina Humana Regular” with a prominent letter “R” indicating “Regular”. This 'R' was mistakenly interpreted as referring to “rabies”. The fact that both the insulin and anti-rabies vaccine vials were multidose contributed to the difficulty distinguishing between them. All personnel associated with the vaccination post were temporarily suspended during the investigation. The case conclusion stated that no veterinarians were involved in the vaccination activities during the campaign. Following the inquiry, the Secretary of Health officially removed the head of the vaccination post in Magé, RJ, from their position. The epidemiological hallmarks of this case series were the clinical signs presented by twenty dogs, along with the two empty insulin vials found among the discarded materials from the vaccination campaign, which raised suspicions of accidental insulin administration. The clinical history states that 20 dogs became severely prostrated and tachypneic; 13 died, and seven underwent necropsy and multiple diagnostic tools. The dog presented for necropsy had comorbidities that corroborated the death. However, none of these comorbidities was considered severe enough to represent a risk to the animal's life. In two dogs, necrotic neurons (37547 and 37548) coincided with high right ventricle blood clot insulin levels. Identifying insulin levels was impossible in one dog with neuronal necrosis (37549). Endogenous insulin half-life is usually short, lasting a few minutes in the bloodstream (Arnolds et al. 2010). The half-life of exogenous insulin is variable between different laboratories and depends on the characteristics of the manufactured molecule. In vitro experiments and postmortem studies show that blood hemolysis directly affects insulin degradation (Wunder et al. 2014; Stephenson et al. 2022), which could directly influence the samples from the submitted dogs. Multiple external and internal factors accelerate autolytic processes (Skowronek 2019). All dogs' carcasses were frozen for five days before the necropsy, but the bodies were presumptively exposed to environmental factors for about 30 minutes before freezing. Although the autolytic processes could have precluded biochemical examination to some degree, we got potassium changes in the vitreous humor in six dogs, and we detected toxic results of insulin by radioimmunoassay (27.82 uUI/ml) at the heart clot sample in one dog. Atomic emission spectroscopy was based on atomization and flame excitation, with essential applications in analyzing metallic elements and wide pathological applications in forensics (Okumura et al. 2004). These flames excite and measure the emission spectrum of various features. These elements are excited in flames at relatively low temperatures to eliminate the excitation of most other metals, providing a range free from interference from other metallic species (Souza 2010). This technique is essential in determining sodium, potassium, and calcium in blood serum, urine, and other biological fluids, such as the vitreous humor (Okumura et al. 2004). The vitreous humor is composed primarily of water, collagen, hyaluronic acid, and electrolytes and is localized in an enclosure with limited exterior contact. The normal vitreous humor potassium concentration in dogs 1 to 4 hours after death is 8.18 ± 0.31 mEq/L (Chavhan et al. 2014). Vitreous humor potassium concentration varied from 7.028 mEq/L to 7.937 mEq/L 30 minutes after death in control dogs used in our study. In all five dogs suspected of insulin poisoning, potassium levels were lower than previously published values (Chavhan et al. 2014) and compared to our study's control dogs. Low potassium concentration in the vitreous humor can indicate a high intracellular potassium influx through an enhanced sodium-potassium-ATPase pump activity. Higher insulin concentrations indirectly positively affect cellular potassium influx due to the stimulation of the sodium-potassium-ATPase pump. High insulin concentrations induce glucose's cellular absorption and, consequently, more ATP production. The sodium-potassium-ATPase pump is an ATP-dependent mechanism, and high concentrations of ATP can stimulate its activity. In the analyzed dogs, low potassium concentrations in the vitreous humor are more likely related to high concentrations of circulating insulin, associated with the histopathological findings of neuronal necrosis, and support the suspicion of insulin poisoning. Three outbreaks of peracute monoethylene glycol poisoning in dogs have been reported in Minas Gerais, southeastern Brazil. The dogs presented polydipsia, pollakiuria, polyuria, vomiting, diarrhea, prostration, and tremors. The lesions were characterized by hydrothorax, moderate pulmonary edema, and hemorrhagic enteritis (Bezerra et al. 2022). Despite the similar peracute presentation, there are marked clinical and pathological distinctions between monoethylene glycol poisoning and hypoglycemia associated with exogenous insulin poisoning. Distemper and rabies were investigated due to the nervous clinical picture of hypoglycemia being similar to acute encephalitis. Both infectious diseases were ruled out by the absence of inflammatory lesions and by negative molecular and immunohistochemistry results tested in the brain samples. The present report documents the human-caused error of swapping vaccine compounds with insulin during a vaccination campaign. Immunization against zoonotic diseases is an essential worldwide strategy with the One Health objective. Rabies is one of the most critical diseases in human history, and the vaccination strategy is the pillar of its eradication in many countries. Although it is essential for human and animal health, vaccination campaigns can have risks. Accidents in vaccination campaigns are poorly documented in the veterinary sciences. Problems can occur in mass vaccination campaigns, like animal accidents (bites, scratches), animal fights, stress-associated problems (hyperthermia, cardiogenic shock), and vaccination side effects due to individual causes or health industrial vaccination mistakes. Vaccination side effects are rare in veterinary medicine and are more associated with particular aspects than fabrication problems (Moore 2010). Application mistakes of vaccines or other medications are common in large animal medicine. Re-utilizing needles and bad hygiene practices are recurrent problems that can lead to bacterial and viral infections from animal to animal (Resende et al. 2020; Quevedo et al. 2011). There have been no reports in veterinary medicine of drugs being mistakenly administered instead of vaccines. Insulin-induced hypoglycemia is rare in small animal practices and is mainly associated with owners' misdosage of insulin in diabetic patients. There are no reported criminal or accidental insulin-induced hypoglycemia cases in nondiabetic animals. Herein, we report an outbreak of accidental insulin-induced hypoglycemia in nondiabetic dogs. All examined dogs had comorbidities that could have corroborated the clinical decline and death; still, none of these conditions were severe enough to represent a direct risk for the lives of each dog. Neuronal necrosis was the most significant lesion related to hypoglycemia in these dogs. As shown, detecting insulin by radioimmunoassay from blood clots is possible. However, positive and negative results should be interpreted with caution due to the degradation and instability of insulin. Atomic emission spectroscopy for postmortem potassium dosage in the vitreous humor of dogs is a promising technique in cases where insulin poisoning is suspected. However, further studies are needed to validate this forensic veterinary science technique. Statements and Declarations The authors have no relevant financial or non-financial interests to disclose. Author Contributions Statement. D.G.U. and L.A.S.T. Conception, L.A.S.T. Wrote original draft, L.A.S.T., G.O.P., R.I.A.C., D.S.L. and D.G.U. Pathological assessment, J.B.P. Clinical assessment, H.A.S. Molecular assessment, A.F. and P.C.M. Toxicological assessment. D.G.U. Project administration. All authors reviewed the final version of the manuscript. Acknowledgments. To the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to grant scholarships, financing code 001. We thank Professor Isabele Angelo and Professor Clayton Gitti from the Department of Epidemiology and Public Health, Federal Rural University of Rio de Janeiro, for indicating the Anatomic Pathology Sector to attend the request from the health department secretary of Magé, RJ. Funding. The authors received no financial support for this article's research, authorship, or publication. Informed consent. The owners of the dogs in this study authorized this clinical and toxicological study. Data availability statement. The entire dataset supporting the results of this outbreak report was published in the article itself. 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Dog identification, weight, clinical evolution, histological findings and results of radioimmunoassay in fatal insulin-induced hypoglycemia Case Number SAP* Record Weight (Kg) Clinical evolution Main histological findings Radioimmunoassay results (serum insulin dosage) (uUI/ml) 1 37544 24.5 Absent data Nonspecific 0.24 2 37545 14 13 h Nonspecific 0.01 3 37546 7.8 3 h Nonspecific 0.01 4 37547 5.1 2 h Temporal cortex: Mild multifocal neuronal necrosis. 11.13 5 37548 16.5 Absent data Frontal and temporal cortex: Moderate multifocal neuronal necrosis. 27.82 6 37549 15 Absent data Brain, frontal cortex: Moderate multifocal neuronal necrosis. 0.01 7 37550 17 Absent data Nonspecific 0.01 *Anatomic Pathology Sector from Federal Rural University of Rio de Janeiro Table 2. Dog analysis of potassium vitreous humor in fatal insulin-induced hypoglycemia compared to control Case Number Analysis of dogs that died from other causes with a 30-minute postmortem interval ( mEq/L ) Dosage of dogs suspected of insulin poisoning ( mEq/L ) 1 7.647805865 4.03780586 2 7.627805865 2.83780586 3 7.427805865 - 4 7.937805865 1.06780586 5 7.027805865 4.67780586 6 - - 7 - 0.74780586 The postmortem potassium reference value in the vitreous humor is 8.18 ± 0.31 mEq/L (Chavhan et al. 2014). - = not detected. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Veterinary Research Communications → Version 1 posted Editorial decision: Revision requested 21 May, 2025 Editor assigned by journal 20 May, 2025 Submission checks completed at journal 20 May, 2025 First submitted to journal 07 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6613595","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":459745459,"identity":"7f06fa74-9641-4e70-8359-3b04613f1834","order_by":0,"name":"Luis Antônio Tondo","email":"","orcid":"","institution":"Louisiana State University","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"Antônio","lastName":"Tondo","suffix":""},{"id":459745469,"identity":"f84ef767-0850-4904-aa60-910921e31695","order_by":1,"name":"Gabriela Oliveira Pereira","email":"","orcid":"","institution":"Federal University of Rio de Janeiro (UFRJ)","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Oliveira","lastName":"Pereira","suffix":""},{"id":459745474,"identity":"086dbadf-2cc2-4ea5-91d2-c242edc6a6ed","order_by":2,"name":"Reina Isabel Cartagena","email":"","orcid":"","institution":"Federal Rural University of Rio de Janeiro (UFRuralRJ)","correspondingAuthor":false,"prefix":"","firstName":"Reina","middleName":"Isabel","lastName":"Cartagena","suffix":""},{"id":459745476,"identity":"03f8293d-fb85-440f-af56-af6dfa2bbd23","order_by":3,"name":"Davi Leal","email":"","orcid":"","institution":"University of Brasília","correspondingAuthor":false,"prefix":"","firstName":"Davi","middleName":"","lastName":"Leal","suffix":""},{"id":459745477,"identity":"40998fd0-ffab-4136-9794-2b96078efa43","order_by":4,"name":"Janice Biazzi 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Paulo","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"César","lastName":"Maiorka","suffix":""},{"id":459745482,"identity":"2662e2c9-f7c0-498d-8297-1683bb314295","order_by":8,"name":"Daniel Guimarães Ubiali","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3PvQrCMBDA8RMhLqVd00Vf4cRBivguFQcXrYIgjulSl4JrBx8m4UAXP1bX4urQ0cHBxFlC3UTy56aDH8cBuFy/mDSzgJhBU8gK2mbFahA0pCFUAb16BAwBTZrem4Cd+IeTkhVS4rdUSsMM5/1UstvaQsJjEqsCacm8kaBZhtFOylb3aCEop0ge0iiDN3ki17+EwkYud6SnIUEpKMqwBrnqK2AI11cadUh4vaPKcbJkvBQqPxuiNl0b8S/TXvVYD5LOdkzVY6UJp31pIx/i6ksAEHwtXC6X6897AaImV6wpxPDWAAAAAElFTkSuQmCC","orcid":"","institution":"Federal Rural University of Rio de Janeiro (UFRuralRJ)","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"Guimarães","lastName":"Ubiali","suffix":""}],"badges":[],"createdAt":"2025-05-07 15:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6613595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6613595/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11259-025-10938-y","type":"published","date":"2025-10-22T16:17:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84662823,"identity":"4b919719-1114-4089-94c1-e57b2ae01674","added_by":"auto","created_at":"2025-06-16 05:07:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2938913,"visible":true,"origin":"","legend":"\u003cp\u003eFatal insulin-induced hypoglycemia in dogs. \u003cstrong\u003ea\u003c/strong\u003e A red dot (local hyperemia) on the left flank's skin indicates the application's location. \u003cstrong\u003eb\u003c/strong\u003eTwo 10 mL empty regular insulin vials were found mixed with other discarded anti-rabies vaccine vials, which resulted in 20 applications of 1 mL in each dog, causing peracute poisoning\u003c/p\u003e","description":"","filename":"Fig1ab.png","url":"https://assets-eu.researchsquare.com/files/rs-6613595/v1/42d8c98415775386d71ca12e.png"},{"id":84662822,"identity":"a85ae1e3-f033-455b-81aa-10477089019d","added_by":"auto","created_at":"2025-06-16 05:07:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4120879,"visible":true,"origin":"","legend":"\u003cp\u003eFatal insulin-induced hypoglycemia in dogs. \u003cstrong\u003ea\u003c/strong\u003e Multiple red and pyknotic nuclei in the temporal telencephalic cortex of Dog 5 indicate neuronal necrosis. Hematoxylin and eosin, Obj. 40x. \u003cstrong\u003eb \u003c/strong\u003eRed perikaryon and pyknotic nucleus in the frontal telencephalic cortex of the Dog 5 indicating neuronal necrosis. Note that at the top right is a single normal neuron. Hematoxylin and eosin, Obj. 63x\u003c/p\u003e","description":"","filename":"Fig2ab.png","url":"https://assets-eu.researchsquare.com/files/rs-6613595/v1/1e31968402b45846ba6ee74d.png"},{"id":94490244,"identity":"7bce6add-3ed5-4df7-86f8-503e0e8b02e2","added_by":"auto","created_at":"2025-10-27 17:08:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7079575,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6613595/v1/11c4a164-7f83-4027-a127-58b168b2b529.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Accidental insulin-induced hypoglycemia in nondiabetic dogs","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eInsulin overdose is a health emergency in dogs and cats. Insulin overdose is more common in cats than in dogs, especially in obese diabetic cats (Whitley et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Insulin is a hormone with great relevance in glucose metabolism. Exogenous insulin therapy is crucial for managing various diseases in small animals. Otherwise, overuse in a patient can lead to severe problems. In the medical literature, cases of insulin poisoning are scarce and primarily associated with accidental misapplication, criminal acts (Bottinelli et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), or suicidal purposes (Wunder et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Even to date, murderers related to insulin application are hard to diagnose. The clinical signs are nonspecific, with few \u003cem\u003epostmortem\u003c/em\u003e and histopathological lesions, and determining the insulin dosage in blood, urine, vitreous humor, or tissue is challenging (Bottinelli et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsulin is an essential hormone that stimulates the uptake and storage of carbohydrates, fatty acids and amino acids (Skowronek \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The sodium-potassium-ATPase pump is an ATP-dependent cellular mechanism that regulates intracellular osmotic concentrations by mediating sodium secretion and potassium influx. Insulin stimulates the cellular uptake of glucose, triggering the activation of sodium-potassium-ATPase pumps, which leads to the influx of extracellular potassium into the cell. Therefore, extracellular potassium and sodium levels could be potential \u003cem\u003epostmortem\u003c/em\u003e biochemical markers for exogenous insulin poisoning, depending on the type of sample selected (Arnolds et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe application of biochemical analysis in \u003cem\u003epostmortem\u003c/em\u003e samples in veterinary medicine is scarce; however, it is broadly used in human forensic medicine. Autolysis and putrefaction directly influence the testing results of \u003cem\u003epostmortem\u003c/em\u003e biochemical techniques. Depending on the condition of the samples, the results can be unreliable, making the analysis difficult to interpret (Madea and Musshoff \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Byard and Tsokos \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lutz et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVitreous humor is a gel-like substance from the posterior chamber of the eye. It is composed primarily of water, collagen, hyaluronic acid, and electrolytes and is kept in a privileged environment compared to other body fluids. Drug penetration into the vitreous humor through the blood-ocular barrier is possible but depends on the drug diffusion rate (Thevis et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Consequently, due to its enclosure within the ocular globe, the vitreous humor maintains low interaction with the environment and is relatively stable and resistant to \u003cem\u003epostmortem\u003c/em\u003e alterations. This feature makes the vitreous humor a promising candidate for toxicological testing (Chavhan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsulin-induced hypoglycemia is rare in small animal practices and is mainly associated with owners\u0026rsquo; misdosage of insulin in diabetic patients (Idowu and Heading \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Herein, we report an outbreak of accidental insulin-induced hypoglycemia in nondiabetic dogs and the utilization of ancillary testing, which could indicate high levels of circulating insulin in animals.\u003c/p\u003e"},{"header":"CASE PRESENTATION","content":"\u003cp\u003e\u003cstrong\u003eEthics.\u0026nbsp;\u003c/strong\u003eWe adopted the CARE Guidelines: Consensus-based clinical case reporting guideline development for transparency of the present case series report. All dog owners authorized the clinicopathological study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmergency care and therapeutics.\u0026nbsp;\u003c/strong\u003eThe health department secretary of Magé, RJ, reported that 20 dogs that attended the same vaccination post showed similar clinical signs.After the vaccination, the dogs were brought to their homes and showed clinical signs of vocalization, shivering, and obtundation. Three dogs had seizures, and one dog vomited. The clinical course of the neurologic signs varied from 2 to 13 hours. One dog was taken to a veterinary clinic for treatment but died immediately after arrival. After the first clinical signs were detected, the dog owners searched for assistance, and the vaccination campaign was blocked. As the health department secretary informed, there was suspicion of accidental parenteral insulin administration in the dogs due to empty insulin vials among the discarded vaccine vials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was reported that twenty dogs presented with peracute clinical signs characterized by intermittent seizures, tremors, inability to stand still, tachypnea, and intense vocalization. In total, 15 dogs that attended the vaccination campaign on the morning of September 4\u003csup\u003eth\u003c/sup\u003e were submitted for clinical service after presenting post-vaccination neurological signs. One of the authors, Janice Biazzi Pires, reported the clinical approach. All dogs were assisted in a private veterinary clinic's emergency and critical care service on the morning of September 4\u003csup\u003eth\u003c/sup\u003e, 2021. The first dog received medical assistance at 9:30 a.m. on September 4\u003csup\u003eth\u003c/sup\u003e. Due to the clinical history of prior vaccination, an anti-anaphylactic shock protocol was initiated. The dog was treated with parenteral adrenaline, venous fluid, and diuretics; however, with an unsuccessful outcome. A similar protocol was applied to 8 dogs, and none survived. In 7 dogs, the blood glucose was assessed and revealed values varying from 19 to 60 mg/dl, which raised concern for hypoglycemia. The clinical protocol was reviewed, and intravenous administration of dextrose associated with sedation was initiated. All dogs submitted to the reviewed clinical approach presented a better outcome and survived.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathology and toxicological sampling.\u0026nbsp;\u003c/strong\u003eThe health department secretary of Magé, RJ, was responsible for sending a picture of two empty regular-acting human insulin vials to the veterinary pathologists as an attachment to the clinical history. Allegedly, 1 mL of regular insulin was administered to twenty dogs due to the dosage recommendation for rabies vaccination. The carcasses of the seven deceased dogs were collected by the team of the health secretary of Magé; however, the dogs' bodies were frozen about 30 minutes after death at the clinic where the emergency care occurred. The dogs' carcasses were kept frozen five days before the necropsy procedure.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeven dog carcasses from different owners were received for necropsy in the \u003cem\u003eSetor de Anatomia Patológica\u003c/em\u003e from the \u003cem\u003eUniversidade Federal Rural do Rio de Janeiro\u003c/em\u003e (SAP/UFRuralRJ). The respective owners informed the history of their dogs. All owners reported that the dogs were sent to an anti-rabies vaccination campaign in Magé, RJ, Brazil.\u003c/p\u003e\n\u003cp\u003eThe seven dogs were submitted to the SAP/UFRuralRJ by the health department secretary of Magé, RJ. They were submitted to a detailed \u003cem\u003epostmortem\u003c/em\u003e examination where material for forensic toxicology was collected for further analysis. All seven dogs were submitted to trichotomy for investigation of injection sites in the skin. A full set of tissues from all seven dogs (brain, spinal cord, lymph nodes, thyroid, adrenal glands, ovaries/testis, as well as samples of omentum, mesentery, gall bladder, uterus, lungs, trachea, esophagus, stomach, intestines, liver, spleen, kidneys, and heart) was collected in 10% buffered formalin solution and processed routinely for histology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor forensic toxicology testing, right ventricle blood clots and vitreous humor were collected from all dogs. The right ventricle clot was submitted for insulin dosage by radioimmunoassay, and the vitreous humor was submitted for a potassium dosage by atomic flame emission photometry to analyze potassium.\u003c/p\u003e\n\u003cp\u003eTo rule out other possible causes of diseases with neurological clinical signs, fresh frontal and parietal cortex, cerebellum, and spinal cord samples were analyzed by qPCR for canine distemper virus (CDV) (Fischer et al. 2013), and formalin-fixed samples by immunohistochemistry for rabies (Pedroso et al. 2008).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOut of the twenty dogs, thirteen died, and five of them did not receive medical aid. Seven dogs were submitted for \u003cem\u003epostmortem\u003c/em\u003e examination. The seven dogs varied from 1 to 14 years old and were two females (one pregnant) and five males. This group had four mixed-breed dogs: one Labrador Retriever, a Basset-hound, and a Poodle. The weight of all animals varied from 5.1 to 24.5kg. None of the dogs had blood glucose values available during the necropsy. At necropsy, the body condition of the seven dogs ranged from 2.5 to 4 (1 to 5 scale). All seven dogs presented a red dot (local hyperemia) on the skin of the left flank, indicating the putative application site (Fig. 1a). During the vaccination campaign, two empty regular-acting human insulin vials and boxes (Fig. 1b) were found mixed with other discarded anti-rabies vaccine vials.\u003c/p\u003e\n\u003cp\u003eAt necropsy, in addition to skin hemorrhage at the site of injection,\u0026nbsp;heterogenic and nonspecific gross lesions were identified in all seven dogs. A grayish irregular vegetative nodule in one dog was attached to the aortic valve (aortic valve endocarditis). Another dog had a red and soft splenic nodule with a 2,5 x 2cm diameter. Also, in this dog, the kidneys were firm and slightly irregular. The pregnant bitch had five well-developed fetuses in the uterus. No other relevant gross lesions were found in the remaining dogs.\u003c/p\u003e\n\u003cp\u003eAt histopathological examination, four dogs had significant amounts of amorphous, homogeneous, and eosinophilic material mixed with foamy macrophages within the pulmonary alveoli (edema). The grossly observed vegetative nodule in the aortic valve was characterized by severe neutrophilic and histiocytic inflammatory infiltrates with moderate basophilic cocci-bacilli colonies (bacterial endocarditis). The splenic nodule corresponded to a fairly demarcated neoplasm composed of pleomorphic endothelial cells (hemangiosarcoma). Two dogs had mild alterations in their kidneys. One had a moderate number of lymphocytes and plasma cells surrounding glomeruli. These glomeruli were hypercellular and had mild basal membrane thickening. The other dog had a moderate number of lymphocytes, plasma cells, and histiocytes expanding the renal interstitium. Three dogs in the frontal and temporal cortex presented a moderate amount of retracted, hypereosinophilic neurons with a hyperchromatic nucleus (neuronal necrosis) (Fig. 2ab).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsulin enzyme radioimmunoassay.\u0026nbsp;\u003c/strong\u003eRight ventricle blood clots from all dogs were collected, kept frozen, and referred to an external laboratory for serum insulin quantification through enzyme radioimmunoassay (Shen et al. 2019). The toxicology examination of the right ventricle clot showed insulin doses in three samples. The values were 27.82 uUI/ml, 11.13 uUI/ml, and 0.24 uUI/ml (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalytical procedure for determination of potassium in vitreous humor.\u003c/strong\u003e Vitreous humor samples from all dogs were collected and referred to forensic toxicology testing. Initially, a calibration curve was made to use the flame photometer (MicroNal brand, model B462). For this purpose, the equipment was zeroed with distilled water, and the suction pressure was adjusted to 0.8 atmospheres. The mother solution was prepared by weighing 50.05g (Gehaka scale BG1000) of potassium chloride and diluted in a 1000 mL volumetric flask, reaching a concentration of 0.05 g/L or 67000 mEq/L. Through this mother solution, dilutions were performed, resulting in six solutions with different concentrations, namely: flask 1 (0.104 mEq/L), flask 2 (1.045 mEq/L), flask 3 (2.094 mEq/L), flask 4 (4.187 mEq/L), flask 5 (8.375), flask 6 (16.755 mEq/L). After building the curve, the analyses were performed. The equipment was reset with distilled water between each analysis to clean it. Reading of analyzed substances: The substances were read from vitreous humor samples of dogs with suspected insulin poisoning and dogs due to natural death with up to 30-minute postmortem intervals. Vials from two dogs (37546 and 37549) had insufficient sample volume for testing. Experimental results: The calibration curve presented a coefficient of determination r2 of 0.9941, showing adequate linearity for the analysis. The chosen range was from 0.1045 mEq/L to 16.75 mEq/L (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative PCR (qPCR) for canine distemper virus (CDV): RNA extraction and cDNA synthesis.\u0026nbsp;\u003c/strong\u003eFresh frontal and parietal cortex, cerebellum, and spinal cord samples from the seven dogs were collected and submitted to RNA extraction employing the TRIzol® Plus RNA Purification Kit according to the manufacturer's instructions. The resulting RNA was treated with DNase I (Invitrogen™, ThermoFisher) to eliminate contaminating genomic DNA. In addition, the quality and integrity of the RNA extracted were evaluated by a Nanodrop spectrophotometer (Invitrogen™, ThermoFisher) and electrophoresis in 1% agarose gel. The RNA extracted was quantified by Qubit™ fluorometer (Invitrogen™, ThermoFisher) using the Qubit™ RNA broad-range assay kit. The complementary DNA (cDNA) was synthesized with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems™, ThermoFisher) according to the manufacturer's instructions. Subsequently, the cDNA was quantified using a Qubit™ fluorometer (Invitrogen™, ThermoFisher), which employed the Qubit™ ssDNA Assay Kit.\u003c/p\u003e\n\u003cp\u003eA combined reverse transcription polymerase chain reaction (RT-qPCR) method was used for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV), as recommended by Fisher et al. (2013). The assay was performed in a MicroAmp® Optical 96-Well Reaction Plate at StepOne Plus thermocycler (Applied Biosystems®, ThermoFisher). The qPCR reaction was carried out in a final volume of 12 µL, containing 1X TaqManTM Universal Master Mix (Applied Biosystems®, ThermoFisher), 0.4 µM of each primer and 0,20 µM of the probe, and 45 ng/µL of cDNA. Thermocycling conditions were 95 °C for 10 minutes, and 40 cycles of denaturation at 95 ºC for 20 s, annealing/polymerization at 60 ºC for 60 s. Samples with Cq values ≤ 40 cycles were considered positive. All dogs were negative for canine distemper virus in real-time polymerase chain reaction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry for rabies.\u003c/strong\u003e 5µm-thick histological sections of the dogs' brainstem were applied to positive slides. Endogenous peroxidase was blocked by incubating the slides in a 3% hydrogen peroxide solution in distilled water for 15 minutes at room temperature. For antigenic recovery, Citrate buffer was used (2.1g of citric acid in 1 liter of distilled water, adjusting the pH to 6.0 with 0.5% NaOH). The sections were treated with 5% skimmed milk (Molico®) diluted in distilled water for 15 minutes. The sections were covered with a solution containing the primary antibody (GeneTex GTX21002 and Biodesign C86307M) in a 1:1,000 dilution in PBS, incubated in a humid chamber for 12 hours at 4 °C (Pedroso et al. 2008). EnVision secondary polymer (Dako, www.agilent.com) was then applied to the sections in an oven at 37 °C for 30 min. Color development was done with 3,3-diaminobenzidine chromogen (DAB + Substrate Chromogen System; DakoCytomation) for 2 min. A bovine rabies case, previously confirmed by direct immunofluorescence (DIF), was used as a positive control. Finally, the sections were counterstained with Harris hematoxylin and coverslipped. Cases were considered positive where structures morphologically compatible with Negri corpuscles were seen within the neuronal cell bodies. All dogs were negative for rabies.\u003c/p\u003e"},{"header":"DISCUSSION AND CONCLUSION","content":"\u003cp\u003eThe dogs presented in this outbreak died due to insulin-induced hypoglycemia, and this highlights the importance of veterinary care during vaccination campaign activities. The clinical and pathological picture was typical of hypoglycemia. Blood glucose measurement is a key diagnostic indicator in cases of suspected insulin poisoning; in the present cases, values ranged from 19 to 60 mg/dL during emergency care, consistent with hypoglycemia and further supported by histopathological findings of neuronal necrosis.\u0026nbsp;The toxicological findings of high levels of \u003cem\u003epostmortem\u003c/em\u003e insulin in the right ventricle blood clot confirm high insulin levels circulating. The high potassium values in the humor vitreous confirm the sodium-potassium pump disturbance. Based on multiple diagnosis tools, we confirm that the dogs went into hypoglycemia associated with exogenous insulin poisoning.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe confusion between the two vials occurred because the commercial insulin vial was labeled “Insulina Humana Regular” with a prominent letter “R” indicating “Regular”. This 'R' was mistakenly interpreted as referring to “rabies”. The fact that both the insulin and anti-rabies vaccine vials were multidose contributed to the difficulty distinguishing between them. All personnel associated with the vaccination post were temporarily suspended during the investigation. The case conclusion stated that no veterinarians were involved in the vaccination activities during the campaign. Following the inquiry, the Secretary of Health officially removed the head of the vaccination post in Magé, RJ, from their position.\u003c/p\u003e\n\u003cp\u003eThe epidemiological hallmarks of this case series were the clinical signs presented by twenty dogs, along with the two empty insulin vials found among the discarded materials from the vaccination campaign, which raised suspicions of accidental insulin administration. The clinical history states that 20 dogs became severely prostrated and tachypneic; 13 died, and seven underwent necropsy and multiple diagnostic tools. The dog presented for necropsy had comorbidities that corroborated the death. However, none of these comorbidities was considered severe enough to represent a risk to the animal's life. In two dogs, necrotic neurons (37547 and 37548) coincided with high right ventricle blood clot insulin levels. Identifying insulin levels was impossible in one dog with neuronal necrosis (37549). Endogenous insulin half-life is usually short, lasting a few minutes in the bloodstream (Arnolds et al. 2010). The half-life of exogenous insulin is variable between different laboratories and depends on the characteristics of the manufactured molecule. In vitro experiments and \u003cem\u003epostmortem\u003c/em\u003e studies show that blood hemolysis directly affects insulin degradation (Wunder et al. 2014; Stephenson et al. 2022), which could directly influence the samples from the submitted dogs. Multiple external and internal factors accelerate autolytic processes (Skowronek 2019). All dogs' carcasses were frozen for five days before the necropsy, but the bodies were presumptively exposed to environmental factors for about 30 minutes before freezing. Although the autolytic processes could have precluded biochemical examination to some degree, we got potassium changes in the vitreous humor in six dogs, and we detected toxic results of insulin by radioimmunoassay (27.82 uUI/ml) at the heart clot sample in one dog.\u003c/p\u003e\n\u003cp\u003eAtomic emission spectroscopy was based on atomization and flame excitation, with essential applications in analyzing metallic elements and wide pathological applications in forensics (Okumura et al. 2004). These flames excite and measure the emission spectrum of various features. These elements are excited in flames at relatively low temperatures to eliminate the excitation of most other metals, providing a range free from interference from other metallic species (Souza 2010). This technique is essential in determining sodium, potassium, and calcium in blood serum, urine, and other biological fluids, such as the vitreous humor (Okumura et al. 2004).\u003c/p\u003e\n\u003cp\u003eThe vitreous humor is composed primarily of water, collagen, hyaluronic acid, and electrolytes and is localized in an enclosure with limited exterior contact. The normal vitreous humor potassium concentration in dogs 1 to 4 hours after death is 8.18\u0026nbsp;±\u0026nbsp;0.31 mEq/L (Chavhan et al. 2014). Vitreous humor potassium concentration varied from 7.028 mEq/L to 7.937 mEq/L 30 minutes after death in control dogs used in our study. In all five dogs suspected of insulin poisoning, potassium levels were lower than previously published values (Chavhan et al. 2014) and compared to our study's control dogs. Low potassium concentration in the vitreous humor can indicate a high intracellular potassium influx through an enhanced sodium-potassium-ATPase pump activity. Higher insulin concentrations indirectly positively affect cellular potassium influx due to the stimulation of the sodium-potassium-ATPase pump. High insulin concentrations induce glucose's cellular absorption and, consequently, more ATP production. The sodium-potassium-ATPase pump is an ATP-dependent mechanism, and high concentrations of ATP can stimulate its activity. In the analyzed dogs, low potassium concentrations in the vitreous humor are more likely related to high concentrations of circulating insulin, associated with the histopathological findings of neuronal necrosis, and support the suspicion of insulin poisoning.\u003c/p\u003e\n\u003cp\u003eThree outbreaks of peracute monoethylene glycol poisoning in dogs have been reported in Minas Gerais, southeastern Brazil. The dogs presented polydipsia, pollakiuria, polyuria, vomiting, diarrhea, prostration, and tremors. The lesions were characterized by hydrothorax, moderate pulmonary edema, and hemorrhagic enteritis (Bezerra et al. 2022). Despite the similar peracute presentation, there are marked clinical and pathological distinctions between monoethylene glycol poisoning and hypoglycemia associated with exogenous insulin poisoning. Distemper and rabies were investigated due to the nervous clinical picture of hypoglycemia being similar to acute encephalitis. Both infectious diseases were ruled out by the absence of inflammatory lesions and by negative molecular and immunohistochemistry results tested in the brain samples.\u003c/p\u003e\n\u003cp\u003eThe present report documents the human-caused error of swapping vaccine compounds with insulin during a vaccination campaign. Immunization against zoonotic diseases is an essential worldwide strategy with the One Health objective. Rabies is one of the most critical diseases in human history, and the vaccination strategy is the pillar of its eradication in many countries. Although it is essential for human and animal health, vaccination campaigns can have risks. Accidents in vaccination campaigns are poorly documented in the veterinary sciences. Problems can occur in mass vaccination campaigns, like animal accidents (bites, scratches), animal fights, stress-associated problems (hyperthermia, cardiogenic shock), and vaccination side effects due to individual causes or health industrial vaccination mistakes. Vaccination side effects are rare in veterinary medicine and are more associated with particular aspects than fabrication problems (Moore 2010). Application mistakes of vaccines or other medications are common in large animal medicine. Re-utilizing needles and bad hygiene practices are recurrent problems that can lead to bacterial and viral infections from animal to animal (Resende et al. 2020; Quevedo et al. 2011). There have been no reports in veterinary medicine of drugs being mistakenly administered instead of vaccines.\u003c/p\u003e\n\u003cp\u003eInsulin-induced hypoglycemia is rare in small animal practices and is mainly associated with owners' misdosage of insulin in diabetic patients. There are no reported criminal or accidental insulin-induced hypoglycemia cases in nondiabetic animals. Herein, we report an outbreak of accidental insulin-induced hypoglycemia in nondiabetic dogs. All examined dogs had comorbidities that could have corroborated the clinical decline and death; still, none of these conditions were severe enough to represent a direct risk for the lives of each dog. Neuronal necrosis was the most significant lesion related to hypoglycemia in these dogs. As shown, detecting insulin by radioimmunoassay from blood clots is possible. However, positive and negative results should be interpreted with caution due to the degradation and instability of insulin. Atomic emission spectroscopy for \u003cem\u003epostmortem\u003c/em\u003e potassium dosage in the vitreous humor of dogs is a promising technique in cases where insulin poisoning is suspected. However, further studies are needed to validate this forensic veterinary science technique.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement.\u003c/strong\u003e D.G.U. and L.A.S.T. Conception, L.A.S.T. Wrote original draft, L.A.S.T., G.O.P., R.I.A.C., D.S.L. and D.G.U. Pathological assessment, J.B.P. Clinical assessment, H.A.S. Molecular assessment, A.F. and P.C.M. Toxicological assessment. D.G.U. Project administration. All authors reviewed the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u003c/strong\u003e To the \u003cem\u003eFunda\u0026ccedil;\u0026atilde;o Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior\u003c/em\u003e (CAPES) to grant scholarships, financing code 001. We thank Professor Isabele Angelo and Professor Clayton Gitti from the Department of Epidemiology and Public Health, Federal Rural University of Rio de Janeiro, for indicating the Anatomic Pathology Sector to attend the request from the health department secretary of Mag\u0026eacute;, RJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThe authors received no financial support for this article\u0026apos;s research, authorship, or publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent.\u003c/strong\u003e The owners of the dogs in this study authorized this clinical and toxicological study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement.\u003c/strong\u003e The entire dataset supporting the results of this outbreak report was published in the article itself. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWhitley NT, Drobatz KJ, Panciera DL (1997) Insulin overdose in dogs and cats: 28 cases (1986-1993). J Am Vet Med Assoc 211:326\u0026ndash;330. \u003c/li\u003e\n\u003cli\u003eBottinelli CN, Cartiser F, B\u0026eacute;valot L, Fanton J (2020) Guitton, Is insulin intoxication still the perfect crime? Analysis and interpretation of \u003cem\u003epostmortem\u003c/em\u003e insulin: review and perspectives in forensic toxicology. Crit Rev Toxicol 50:324\u0026ndash;347. https://doi.org/10.1080/10408444.2020.1762540\u003c/li\u003e\n\u003cli\u003eWunder C, Kauert GF, Toennes S W (2014) Factors leading to the degradation/loss of insulin in postmortem blood samples. Forensic Sci Int 241:173\u0026ndash;177. https://doi.org/10.1016/j.forsciint.2014.06.003\u003c/li\u003e\n\u003cli\u003eSkowronek R (2019) Insulin in Forensic Medicine and Toxicology. Intech Open. https://doi.org/10.5772/intechopen.76691\u003c/li\u003e\n\u003cli\u003eMadea B, Musshoff F (2007) \u003cem\u003ePostmortem\u003c/em\u003e biochemistry. Forensic Sci Int 165:165\u0026ndash;171. https://doi.org/10.1016/j.forsciint.2006.05.023\u003c/li\u003e\n\u003cli\u003eByard RW, Tsokos M (2013) The challenges presented by decomposition. Forensic Sci Med Pathol 9\u003cem\u003e:\u003c/em\u003e135\u0026ndash;137. https://doi.org/10.1007/s12024-012-9386-2\u003c/li\u003e\n\u003cli\u003eLutz R, Pedal I, Wetzel C, Mattern R (1997) Insulin injection sites: morphology and immunohistochemistry. Forensic Sci Int 90:93\u0026ndash;101.https://doi.org/10.1016/s0379-0738(97)00153-9\u003c/li\u003e\n\u003cli\u003eThevis M, Thomas A, Sch\u0026auml;nzer W, Ostman P, Ojanper\u0026auml; I (2012) Ojanper\u0026auml;, Measuring insulin in human vitreous humour using LC-MS/MS. Drug Test Anal 4:53\u0026ndash;56. https://doi.org/10.1002/dta.368\u003c/li\u003e\n\u003cli\u003eIdowu O, Heading K (2018) Hypoglycemia in dogs: Causes, management, and diagnosis. Can Vet J 59:642\u0026ndash;649. PMCID: PMC5949948\u003c/li\u003e\n\u003cli\u003eFischer CD, Ikuta N, Canal CW, Makiejczuk A, Allgayer MC, Cardoso CH, Lehmann FK, Fonseca AS, Lunge VR (2013) Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis. J Virol Methods 194: 39\u0026ndash;45. https://doi.org/10.1016/j.jviromet.2013.08.002\u003c/li\u003e\n\u003cli\u003ePedroso PMO, Pescador CA, Bandarra PM, Raymundo DL, Borba MR, Wouters F, Bezerra Jr PS, Driemeier D (2008) Padroniza\u0026ccedil;\u0026atilde;o da t\u0026eacute;cnica de imuno-histoqu\u0026iacute;mica para raiva em amostras de tecido do sistema nervoso central de bovinos fixadas em formol e emblocadas em parafina. Pesq Vet Bras 28:627\u0026ndash;632. https://doi.org/10.1590/S0100-736X2008001200012\u003c/li\u003e\n\u003cli\u003eArnolds S, Kuglin B, Kapitza C, Heise T (2010) How pharmacokinetic and pharmacodynamic principles pave the way for optimal basal insulin therapy in type 2 diabetes. Int J Clin Pract 64:1415\u0026ndash;1424. https://doi.org/10.1111/j.1742-1241.2010.02470.x\u003c/li\u003e\n\u003cli\u003eStephenson L, Van den Heuvel C, Humphries M, Byard R W (2022) Characteristics of fatal insulin overdoses. Forensic Sci Med Pathol 18:429\u0026ndash;441. https://doi.org/10.1007/s12024-022-00511-3\u003c/li\u003e\n\u003cli\u003eZhou C, Byard RW (2011) Factors and processes causing accelerated decomposition in human cadavers - An overview. J. Forensic Leg Med 18:6\u0026ndash;9. https://doi.org/10.1016/j.jflm.2010.10.003\u003c/li\u003e\n\u003cli\u003eOkumura F, Cavalheiro \u0026Eacute;TG, N\u0026oacute;brega JA (2004) Experimentos simples usando fotometria de chama para ensino de princ\u0026iacute;pios de espectrometria at\u0026ocirc;mica em cursos de qu\u0026iacute;mica anal\u0026iacute;tica. Qu\u0026iacute;mica Nova 27:832\u0026ndash;836. https://doi.org/10.1590/S0100-40422004000500026\u003c/li\u003e\n\u003cli\u003eSouza JEG (2010) An\u0026aacute;lise Qu\u0026iacute;mica Instrumental - Fundamentos da fotometria de chama. UNICAP, Recife, P18\u003c/li\u003e\n\u003cli\u003eChavhan SG, Balachandrra C, Nambi AP, Dhinakar Raj G, Vairamuthu SA (2014) Study on vitreous humor between-eye differences and baseline values of potassium, calcium, sodium and glucose immediately after death in dogs. Ind J Vet Anim Sci Res 43:372\u0026ndash;375. \u003c/li\u003e\n\u003cli\u003eBezerra L S, Amaral C I, Moreira L G A, Freitas D C O, Pierezan F (2022) Pesq Vet Bras 42(Supl.):89\u0026ndash;90. https://pvb.com.br/portal/download_artigo/MzQwOHwyMDI1MDQxNjA5MDAyNA==\u003c/li\u003e\n\u003cli\u003eMoore GE, (2010) HogenEsch H Adverse vaccinal events in dogs and cats. Vet Clin North Am Small Anim Pract 40:\u003cem\u003e \u003c/em\u003e393\u0026ndash;407. https://doi.org/10.1016/j.cvsm.2010.02.002\u003c/li\u003e\n\u003cli\u003eResende CF, Galinari GCF, Victor RM, Kassar TC, Arcebispo TLM, Delarmelina E, Leite RC, Reis JKP (2020) Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leucosis. Pesq Vet Bras 40 (12):977\u0026ndash;984. https://doi.org/10.1590/1678-5150-PVB-6731\u003c/li\u003e\n\u003cli\u003eQuevedo PS, Ladeira SRL, Soares MP, Marcolongo-Pereira C, Sallis ESV, Grecco FB, Estima-Silva P, Schild AL (2011) Tetanus in cattle in southern Brazil: study of 24 outbreaks. Pesq Vet Bras 31(12):1066\u0026ndash;1070. https://doi.org/10.1590/S0100-736X2011001200005\u003c/li\u003e\n\u003cli\u003eShen Y, Prinyawiwatkul W, Xu Z (2019) Insulin: a review of analytical methods. Analyst 144(14):4139\u0026ndash;4148. https://doi.org/\u003cu\u003e10.1039/c9an00112c.\u003c/u\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"822\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"bottom\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eDog identification, weight, clinical evolution, histological findings and results of radioimmunoassay in fatal insulin-induced hypoglycemia\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase Number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSAP*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRecord\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeight\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClinical evolution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMain histological findings\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRadioimmunoassay results\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(serum insulin dosage) (uUI/ml)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e37544\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e24.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003eAbsent data\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003eNonspecific\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e37545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003e13 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003eNonspecific\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e37546\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003e3 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003eNonspecific\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e37547\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003e2 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003eTemporal cortex: Mild multifocal neuronal necrosis.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e11.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e37548\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e16.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003eAbsent data\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003eFrontal and temporal cortex: Moderate multifocal neuronal necrosis.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e27.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e37549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003eAbsent data\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003eBrain, frontal cortex: Moderate multifocal neuronal necrosis.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 7.05596%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6.93431%;\"\u003e\n \u003cp\u003e37550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5.71776%;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.12409%;\"\u003e\n \u003cp\u003eAbsent data\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.3625%;\"\u003e\n \u003cp\u003eNonspecific\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 29.8054%;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" style=\"width: 100%;\"\u003e\n \u003cp\u003e*Anatomic Pathology Sector from Federal Rural University of Rio de Janeiro\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"455\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eDog analysis of potassium vitreous humor in fatal insulin-induced hypoglycemia compared to control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCase Number\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalysis of dogs that died from other causes with a 30-minute \u003cem\u003epostmortem\u003c/em\u003e interval (\u003c/strong\u003e\u003cstrong\u003emEq/L\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDosage of dogs suspected of insulin poisoning (\u003c/strong\u003e\u003cstrong\u003emEq/L\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e7.647805865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e4.03780586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e7.627805865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e2.83780586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e7.427805865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e7.937805865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e1.06780586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e7.027805865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e4.67780586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.967%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.956%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43.0769%;\"\u003e\n \u003cp\u003e0.74780586\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u003cem\u003eThe postmortem\u003c/em\u003e potassium reference value in the vitreous humor is 8.18 \u0026plusmn; 0.31 mEq/L (Chavhan et al. 2014). - = not detected.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Veterinary pathology, neuropathology, emergency care, insulin poisoning, humor vitreous, potassium","lastPublishedDoi":"10.21203/rs.3.rs-6613595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6613595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e Herein, animal cases of insulin-induced hypoglycemia are presented in nondiabetic patients. We report an outbreak of iatrogenic insulin poisoning in dogs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Twenty dogs presented peracute clinical signs, and seven were submitted for \u003cem\u003epostmortem\u003c/em\u003e and histological examination in the \u003cem\u003eSetor de Anatomia Patológica\u003c/em\u003e (SAP) at the Federal Rural University of Rio de Janeiro, Brazil. Blood serum samples from the cardiac clot of the right ventricle of the dogs were sent for radioimmunoassay examination for insulin dosage. Vitreous humor samples were submitted for flame atomic emission photometry for potassium level determination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e All dogs had previously attended an anti-rabies vaccination campaign on the same day and time and showed peracute clinical signs after the application. The respondents reported suspicions about accidental parenteral administration of insulin in the dogs. Tremors, inability to stand still, tachypnea, and intense vocalization were evident in 2 to 13 hours of clinical evolution. Histologically, there was neuronal necrosis in the temporal and frontal cortex. Radioimmunoassay identified insulin in three of the submitted samples. Flame atomic emission photometry revealed high levels of potassium in all samples. The epidemiological, clinical, and histopathological findings associated with identifying insulin in the blood serum and atomic emission spectroscopy for potassium recovery endorsed the suspicion of insulin poisoning.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e For the first time in veterinary medicine, accidental insulin-induced hypoglycemia is reported in nondiabetic dogs. Flame atomic emission photometry was a promising tool for indicating high circulating insulin levels in forensic veterinary medicine.\u003c/p\u003e","manuscriptTitle":"Accidental insulin-induced hypoglycemia in nondiabetic dogs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 05:07:33","doi":"10.21203/rs.3.rs-6613595/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-21T08:06:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-21T01:59:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-21T01:59:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2025-05-07T15:41:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d397d2e7-f4a9-451b-aaf8-da365f59d3e9","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:24:23+00:00","versionOfRecord":{"articleIdentity":"rs-6613595","link":"https://doi.org/10.1007/s11259-025-10938-y","journal":{"identity":"veterinary-research-communications","isVorOnly":false,"title":"Veterinary Research Communications"},"publishedOn":"2025-10-22 16:17:20","publishedOnDateReadable":"October 22nd, 2025"},"versionCreatedAt":"2025-06-16 05:07:33","video":"","vorDoi":"10.1007/s11259-025-10938-y","vorDoiUrl":"https://doi.org/10.1007/s11259-025-10938-y","workflowStages":[]},"version":"v1","identity":"rs-6613595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6613595","identity":"rs-6613595","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}