Perivascular Cuffing Research Articles

Enhanced glycolytic metabolism supports transmigration of brain-infiltrating macrophages in multiple sclerosis.

The migration of leukocytes into the CNS drives the neuropathology of multiple sclerosis (MS). This penetration likely utilizes energy resources that remain to be defined. Using the experimental autoimmune encephalomyelitis (EAE) model of MS, we determined that macrophages within the perivascular cuff of post-capillary venules are highly glycolytic as manifested by strong expression of lactate dehydrogenase A (LDHA) that converts pyruvate to lactate. These macrophages expressed prominent levels of monocarboxylate transporter-4 (MCT-4) specialized in secreting lactate from glycolytic cells. The functional relevance of glycolysis was confirmed by siRNA-mediated knockdown of LDHA and MCT-4, which decreased lactate secretion and macrophage transmigration. MCT-4 was in turn regulated by EMMPRIN (CD147) as determined through co-expression/co-immunoprecipitation studies, and siRNA-mediated EMMPRIN silencing. The functional relevance of MCT-4/EMMPRIN interaction was affirmed by lower macrophage transmigration in culture using the MCT-4 inhibitor, α-cyano-4-hydroxy-cinnamic acid (CHCA), a cinnamon derivative. CHCA also reduced leukocyte infiltration and the clinical severity of EAE. Relevance to MS was corroborated by the strong expression of MCT-4, EMMPRIN and LDHA in perivascular macrophages in MS brains. These results detail the metabolism of macrophages for transmigration from perivascular cuffs into the CNS parenchyma and identifies CHCA and diet as potential modulators of neuro-inflammation in MS.

Intragastric and intracoelomic injection challenge models of tilapia lake virus infection in Nile tilapia (Oreochromis niloticus L.) fingerlings.

To gain a better understanding of the pathogenesis of tilapia lake virus (TiLV) infections in Nile tilapia (Oreochromis niloticus), fingerlings were challenged with a single dose of 1×104 TCID50 /fish of TiLV utilizing intracoelomic/intraperitoneal (ICch ) or intragastric (IGch ) routes. Acute mortalities were present in both groups, reaching 70 and 40% in ICch and IGch after 10days, respectively. Challenged fish presented erratic swimming, lethargy, anorexia, exophthalmia and cutaneous petechiae and ecchymoses. Histological changes in challenged groups included syncytial formation, intracytoplasmic inclusion bodies and multifocal hepatocellular degeneration and necrosis. In addition, multifocal areas of mild proliferation of glial cells and lymphocytic perivascular cuffing were observed in the brain of exposed challenged groups. TiLV RNA was detected in gills and faeces of challenged fish using quantitative reverse transcriptase-PCR, as well as in the tank water holding challenged fish. Moreover, TiLV RNA was detected in scrolls obtained from formalin-fixed paraffin-embedded tissue blocks from challenged fish. Results from this study suggest that IG methods represent an additional method to study the pathogenesis of the disease in this species, as it results in infection and diseases as in naturally occurring cases and does not bypass important mucosal immune responses as injectable routes do.

Histopathological lesions in reproductive organs, distal spinal cord and peripheral nerves of horses naturally infected with Trypanosoma equiperdum

BackgroundDourine, a venereal transmitted trypanosomosis caused by Trypanosoma equiperdum, has different clinical signs related to the reproductive and nervous system. Pathologic tissue changes associated with the disease are poorly described. The present study describes the histopathological lesions in naturally T. equiperdum-infected horses in the chronical stage of dourine.ResultsFour chronically dourine diseased horses underwent a post-mortem examination. They were Woo test negative, but CATT/T. evansi positive, had a low packed cell volume (PCV) and exhibited obvious clinical signs of dourine. Post-mortem examination did not reveal gross lesions in the organs assumed to be responsible for the symptomatology. On histopathology, genital organs were affected, with mononuclear cell infiltration and erosions and degeneration of seminiferous tubules and perivascular lymphoplasmacytic cuffing in the uterus. In the nervous system, mononuclear cell infiltration was located in peripheral nerves, ganglia and in the spinal cord, leading to axonal degeneration. Real-time PCR using ITS primer revealed the presence of trypanosomes in these organs and conventional PCRs using maxicircle and RoTat1.2 primers further confirmed the involvement of T. equiperdum since the DNAs from the vagina, testicle, distal spinal cord, sciatic and obturator nerves found to be positive for maxicircle and negative for RoTat 1.2.ConclusionsThe histopathological lesions in the spinal cord and peripheral nerves explain the incoordination of the hind legs in T. equiperdum-infected horses, whilst its presence in the genital tract exemplifies the venereal transmission.

Patterns of immune-cell infiltration in murine models of melanoma: roles of antigen and tissue site in creating inflamed tumors.

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/β-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/β-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.

Nervous form of listeriosis in buffaloes

ABSTRACT: Listeriosis is a disease that affects several animal species, including humans, and has three different forms of presentation: encephalic, reproductive, or septicemic. The nervous form is caused mainly by the bacterium Listeria monocytogenes. In Brazil, this disease has already been described in sheep, goats, and cattle. There are no reports of the disease in buffaloes in Brazil and worldwide. The objective of this study was to describe an outbreak of listeric meningoencephalitis in buffaloes in the state of Pará, Brazil. The outbreak occurred in a property located in the municipality of Bujaru, in the eastern Amazon, from May to July 2016. In a herd of 47 buffaloes, three animals (Cases 1, 2 and 3), aged <40 days, presented a neurological condition with locomotion difficulty characterized by paralysis of the four limbs, hypoesthesia, lateral recumbency, and death. Morbidity was 6.38% and lethality was 100%. At necropsy, no significant macroscopic lesions were found. Samples of the central nervous system were collected, fixed in 10% buffered formalin, and routinely processed for histopathological analysis. The main microscopic changes observed were unilateral microabscesses in the brainstem composed predominantly of mononuclear cells, with fewer polymorphonuclear cells, and perivascular cuffs composed mostly of mononuclear cells and few neutrophils. Samples of Cases 1 and 2 revealed Gram-positive bacteria in the areas of necrosis by the Gram’s stain technique. Samples of Case 1 were positive in immunohistochemistry for L. monocytogenes. Diagnosis of the nervous form of listeriosis was based on epidemiological data, clinical profile, and immunostaining for Listeria monocytogenes. Results showed that listeriosis should be considered in the differential diagnosis in buffaloes with nervous signs.

Molecular characterization and histopathological studies on Fasciola gigantica in Mithun (Bos frontalis)

Mitochondrial DNA sequence of the sub-unit 1 of cytochrome c oxidase (Cox1) and ribosomal DNA sequence of the first internal transcribed spacer (ITS-1) of Fasciola collected from mithun and cattle from Arunachal Pradesh, India were characterized at molecular level in order to identify the species of parasite. Based on sequence and phylogenetic analysis, the identity of the parasite was confirmed as Fasciola gigantica in mithun as well as from cattle. In order to know histopathological alteration in Fasciola infection, a histopathological study was performed on eight liver specimens, out of 110 animals studied during the period from 2010-2016. Histopathological examination of F.gigantica infected liver showed extensive fibrous connective tissue proliferation with necrosis of hepatocytes and infiltration of polymorphonuclear cell. There was evidence of migratory tracts of parasites with losing of normal lobular hepatic architecture. Bile duct proliferation was followed by congestion of portal vein with perivascular cuffing with surrounding degeneration of hepatocytes. The hepatocytes showed pyknosis with hyperplasia of bile duct.

Safety, Diagnostic Accuracy, and Effects of Endoscopic Ultrasound Fine-Needle Aspiration on Detection of Extravascular Migratory Metastases

Tumor cells can migrate via diminutive perivascular cuffing to distant sites along blood vessels to form extravascular migratory metastases (EVMM). These metastases usually areidentified during surgery or autopsies. We aimed to evaluate the feasibility and safety ofendoscopic ultrasound fine-needle aspiration (EUS-FNA) of perivascular soft-tissue cuffs todetect EVMM. We compared findings from EUS with those from noninvasive cross-sectionalimaging (reference standard) of patients who underwent EUS-FNA to assess suspected EVMM and studied the effects on pancreatic tumor staging and determination of resectability. We performed a retrospective analysis of 253 patients (mean age, 62 ± 12 y) who underwent EUS-FNA of 267 vessels for evaluation of suspected EVMM, from April 2001 through May 2018. EUS findings were compared with those from computed tomography (CT) and magnetic resonance imaging (MRI) as the reference standard. Lesions were considered to be malignant based on cytology analysis of FNA samples, histology analyses of surgical or biopsy specimens, or vascular abnormalities detected by CT or MRI that clearly indicate EVMM. Thirty patients were found to have benign lesions. The remaining 223 patients who had malignancies (166 with pancreatic ductal adenocarcinomas [PDACs]), underwent further analyses. A median of 4 FNAs (range, 1-20 FNAs) were obtained from 4-mm perivascular soft-tissue cuffs (range, 2-20 mm). FNA and cytology analysis showed malignant cells in 163 vessels (69.4%) from 157 patients (70.4%). CT or MRI did not detect EVMM in 44 patients (28%) with malignancies, including 24 patients (24%) with newly diagnosed PDAC. Detection of EVMM by EUS-FNA resulted in upstaging of 15 patients and conversion of 14 patients with PDAC from resectable (based on CT or MRI) to unresectable. No adverse events were reported during a follow-up period of 3.9 months (range, 0-117 mo). EUS-FNA and cytologic analysis of perivascular soft-tissue cuffs can detect EVMM that were not found in 28% of patients by CT or MRI. Detection of EVMM affects tumor staging and determination of tumor resectability.

AMPK Activation Improves Survivability after Toxic Gas Exposure

IntroductionInhalation injury caused by irritant gases such as smoke, ozone, chlorine and bromine (Br2) results in an environmental and occupational hazards that leads to reactive airway disease syndrome (RADS), acute lung injury (ALI), and even death from respiratory failure. Development of interventional therapeutic agents, which when given post exposure, prevent acute inhalation injury and decrease mortality are urgently needed. AMP‐activated protein kinase (AMPK) is a metabolic sensor that maintains cellular ATP levels during metabolic stress. AMPK also acts independently of AMP and enhances vascular integrity by preventing actin stress fiber formation and limiting changes in cell morphology necessary for increased endothelial permeability. Here in the current study we tested the hypothesis that a single injection of an AMPK activator, such as 5‐aminoimidazole‐4‐carboxamide‐1‐beta‐D‐ribofuranoside (AICAR), administered intraperitoneally to C57BL/6 mice, post exposure to a lethal dose of inhaled Br2, decreases lung injury and mortality.MethodsC57BL/6 mice were exposed to Br2 (600 ppm, 45min) in environmental glass chambers and then returned to room air. Control mice breathed ambient air throughout this protocol. Six hours after exposure to Br2, mice were treated with either AICAR (10 mg/kg BW, intraperitoneally) or vehicle (Earle's balanced salt solution. Next seven days mice were monitored at interval of six hours. In another set of experiments, mice were sacrificed 24 hours post Br2 inhalation and were either lavaged with one ml of normal saline for protein and inflammatory cell counts or their lungs were fixed by instillation of formalin at 25 cm H20; and processed for histology.ResultsThe analysis of the Kaplan Meier survival curve showed that Br2 exposed mice developed progressive respiratory distress. Median survival after Br2 exposure was one day. However, mice receiving AICAR had a median survival of six days. Exposure to Br2 caused significant increase in total protein and number of inflammatory cells in BALF at 24 hours post exposure, consistent with severe injury to the blood gas barrier. AICAR returned these values to their corresponding air controls. Br2 exposure significantly reduced the level of AMPK phosphorylation whereas AICAR treatment brought its level to normal in animal lungs. Lung H&E staining showed that AICAR attenuated Br2‐induced lung edema, alveolar congestion, septal thickening, neutrophil infiltration, hyaline membrane and perivascular cuffing. Results also showed that AICAR enhanced autophagy, which was significantly reduced after Br2 exposure suggesting a possible mechanism of AICAR‐mediated cyto‐protection.ConclusionsIn conclusion data suggest that activation of lung AMPK might be a useful target for a therapeutic intervention to reverse Br2‐induced acute lung injury.Support or Funding InformationThis study was supported by 1U01ES027697‐01 and 5U01ES026458‐02 to S. Matalon.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Brain Infection by Hepatitis E Virus Probably via Damage of the Blood-Brain Barrier Due to Alterations of Tight Junction Proteins.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

Systemic Toxoplasmosis in Kittens

Background: Toxoplasmosis is a worldwide disease that affects virtually all species of warm-blooded animals. The felids, domestic and wild, are considered the definitive hosts of the protozoan. In Brazil, Toxoplasma gondii infection has been diagnosed in horses, goats, primates, dogs and cats. In the backlands of Paraíba, the disease has been sporadically reported affecting dogs with canine distemper and swine, but cases of systemic toxoplasmosis in cats have not yet been described. The aim of the present study was to describe the main epidemiological, clinical and anatomopathological aspects of two cases of systemic toxoplasmosis in kittens.Cases: Two kittens were affected with three (cat 1) and six (cat 2) months old, females, and crossbreed. The kittens had not been vaccinated or dewormed, and were raised with other cats in a peridomiciliary regime in a rural area in the backlands of Paraíba. Cat 1 was thin, apathetic, dehydrated, tachypneic and with pale mucous membranes. Cat 2, showed inappetence, apathy, jaundice, fever, dehydration, dyspnea and abdominal breathing pattern. At necropsy, non-collapsed, shiny, reddish lungs with multifocal whitish areas, punctuated or nodular, measuring from 0.1 to 0.3 cm in diameter, were found on the pleural surface and parenchyma. The livers were pale, with lobular pattern accentuation, and reddish depressed multifocal areas randomly distributed on the capsular surface. Histologically, multifocal to coalescent areas of necrosis, moderate (cat 1) or marked (cat 2), associated with intralesional bradyzoites and tachyzoites and variable lymphoplasmacytic infiltrate were observed. In cat 2, numerous bradyzoites were visualized in the gray matter of the left cerebral hemisphere (temporal and parietal lobes), sometimes associated with a moderate lymphoplasmacytic inflammatory infiltrate. In the perivascular spaces of the cerebral cortex, discrete inflammatory lymphoplasmacytic infiltrate (perivascular cuffs) were observed. Immunohistochemistry for T. gondii revealed strong immunolabelling of the parasitic organisms.Discussion: The diagnosis of systemic toxoplasmosis was established based on anatomopathological findings and histomorphological characteristics of the agent, being confirmed by immunohistochemistry. The systemic disease occurs most often in young animals, especially immunocompromised or immunologically immature neonates. In these kittens, it was not possible to determine the presence of an intercurrent immunosuppressive condition; however, both animals were kept in the peridomiciliary regime and were in contact with other cats, which may have acted as a source of infection. Besides, both kittens were raised in rural environments, condition that associated with the usual predatory behavior of this species may have favored the ingestion of infected intermediate hosts. The clinical signs of the disease vary considerably depending on the location and severity of the lesions. Infection in cats usually is asymptomatic or promotes self-limiting diarrhea as a result of protozoal enteroepithelial replication; but in some cases the protozoan can spread to other tissues, determining the occurrence of necrotic processes, dysfunction and clinical manifestations. Systemic toxoplasmosis occurs sporadically in kittens in the backlands of Paraíba, with pulmonary and hepatic clinical manifestations. The histopathological findings are characterized by tissue necrosis and lymphoplasmacytic inflammation associated with intralesional protozoa. The diagnosis can be established based on the histomorphologic characteristics of the agent and confirmed by immunohistochemistry.

African Horse Sickness virus: pathogenicity in an IFNAR(-/-) mouse model of infection

African Horse Sickness (AHS) is a highly lethal, vector-borne viral disease of equids, endemic to sub-Saharan Africa but with a history of outbreaks into Europe. The pathogenesis of the virus is not fully understood; some studies have shown that certain proteins are linked to virulence, and that the outcome of infection is highly dependent on viral factors. Disease can also manifest with several different presentations in the equine host (fever form, cardiac form, pulmonary form and mixed form) but the mechanism underlying this variation is not fully understood. Pathogenicity and virulence studies of AHSV are difficult to perform in horses for logistical, ethical and financial reasons. As an alternative, Interferon-alpha receptor knockout (IFNAR-/-) mice have previously been used in AHSV vaccinology studies. Full pathology characterization of the AHSV infection in this model is the primary objective of our work, which will address questions regarding pathogenesis of AHSV. Here we present data collected from experimental infection of IFNAR (-/-) mice with a strain of AHSV serotype 4, including: clinical signs; histopathology; immuno-histochemical analysis of immune response to infection; antigen detection in tissues; and transmission electron microscopy of AHSV infected tissues. In addition, we will show data from experimental infection in this animal model comparing the pathogenicity of different AHSV strains. Results obtained indicate AHSV-4 infection is correlated with oedema and pneumonia in the lungs, inflammation in the liver and meningitis plus perivascular cuffing in the cerebrum. Other data shows that different strains of AHSV differ in terms of their pathogenicity and tropism.

Zika virus: out of sight but not out of the mind. Viral persistence and host responses within the central and peripheral nervous systems

Following the rapid spread of Zika virus infection through the Caribbean and Americas, model systems of Zika virus infection have been established to determine disease pathology and potential health impacts for those infected with this neurotropic flavivirus. NIBSC has established both Old World (rhesus and cynomolgus macaques) and New World (red-bellied tamarins) non-human primate disease models of human Zika virus infection. Animals were infected sub-cutaneously with the Caribbean Zika isolate PRVABC59 and pairs terminated during primary viremia (3 dpc) or post clearance of plasma viremia (42 and 101 dpc). FFPE sections from multiple tissues, including brain and peripheral nerves were analysed for Zika virus RNA (RNAscope), viral proteins and host responses (immunohistochemistry). All animals became infected and rapidly cleared high levels of peripheral viremia. Zika virus RNA was detected in multiple tissues from 3 dpc with low levels of viral RNA and viral NS1 protein remaining detectable through to 101 dpc. Viral RNA was associated with both glial and neuronal cells throughout the brain and schwann cell nuclei in peripheral nerves. Neuroinflammation (astrogliosis, microgliosis, peri-vascular cuffing) and increased levels of CD3+, CD8+ or CD45+ cells were present from 3 dpc through to 101 dpc. Disruption of MAP2 neuronal dendrite staining and peripheral nerve myelin basic protein staining was also observed. The continued detection of Zika virus within tissues 3 months post infection and in the absence of a detectable peripheral viremia raises questions regarding potential long term effects of this virus and associated inflammatory responses within the nervous systems.

Anatomopathological and Immunohistochemical Apects of Distemper Virus in Crab-Eating-Foxes and Pampa-Foxes

Background: Distemper is a contagious disease with worldwide distribution, which is caused by a single-stranded RNA virus of the genus Morbillivirus. The Crab-eating fox (Cerdocyon thous) and the Pampas fox (Lycalopex gymnocercus), wild canids commonly found in the state of Rio Grande do Sul, are highly susceptible to the disease. Distemper virus infection often shows a multisystemic presentation. Central nervous system lesions are frequently reported, and may lead to death of wild canids. Distemper virus infection affecting these species has been cited by several studies; nonetheless, case descriptions involving the Crab-eating fox and the Pampas fox are infrequent. Therefore, the objective of the present study is to describe the anatomopathological and immunohistochemical aspects of distemper in the referred species, aiming to assist with the diagnosis of the disease.Materials, Methods & Results: A retrospective study was performed, and the necropsy reports of Crab-eating foxes and Pampas foxes admitted to SPV-UFRGS from 2010 to 2016 were reviewed. Necropsy reports were revised in order to obtain information related to the clinical history and anatomopathological findings, and cases compatible with distemper were selected. Data regarding the affected species, sex, approximate age, origin and clinical signs were compiled and analyzed. Selected histological sections of brain and cerebellum were submitted for immunohistochemistry (IHC) for monoclonal antibody canine distemper virus. In the studied period, 20 animals of the referred species were subjected to necropsy, of which four were diagnosed with distemper as a cause of death. Of the affected animals, two were female and two were male. Three were juvenile and one was an adult animal. Clinical signs reported included myoclonia, opisthotonos, forelimb paresis and nystagmus. No significant gross changes were noted. Histopathological findings were restricted to the central nervous system, especially in the cerebellum, and the main lesion was demyelination, as well as gemistocytic astrocytes, malacia with Gitter cells, gliosis and perivascular cuffing. Inclusion bodies were observed in only one case. Diffuse and marked viral antigen positive immunostaining was detected, mainly in the cytoplasm of astrocytes, predominantly in the cerebellum. Discussion: The diagnosis of distemper affecting the Crab-eating fox and Pampas fox was based on the clinical and histopathological findings, along with viral antigen identification through immunohistochemistry. Clinical signs were restricted to the central nervous system and were associated with the affected area. Significant gross changes in the central nervous system were not observed, similarly to what has been described by other authors. The main histological lesion was demyelination in the cerebellum, which is in agreement with previous reports in wild canids. Similarly to the findings reported in dogs and mustelids, positive immunostaining was observed mainly in the cytoplasm of astrocytes in the cerebellum; however, in our cases diffuse marked staining was noted. Although some authors have reported that viral antigens disappear in demyelinating lesions, due to the inflammation associated with the host immune response, the opposite was observed in the present study, suggesting viral persistency linked to a weak immune response in these species. The clinical and pathological changes observed were compatible with the neurologic phase of distemper; therefore, it is important to include this agent among the differential diagnosis in the referred wild canids, mainly in animals presenting neurological signs. Immunohistochemichal evaluation was important to the establishment of postmortem diagnosis of distemper, since characteristic viral inclusion bodies were rarely observed through histopathology.

Lesions and Distribution of Viral Antigen in the Brain of Hamsters Infected With Equine Herpesvirus (EHV)-9, EHV-1 Strain Ab4p, and Zebra-Borne EHV-1.

Encephalitis in hamsters, which was induced by equine herpesvirus (EHV)-9, EHV-1 strain Ab4p, and zebra-borne EHV-1, was investigated and compared to assess viral kinetics and identify the progression and severity of neuropathological findings. Hamsters were inoculated with EHV-9, EHV-1 strain Ab4p, and zebra-borne EHV-1 via the nasal route and euthanized at 24, 48, 72, 96, 120, 144, and 168 hours postinoculation (HPI). The inoculated hamsters had mild to severe neurological signs at 60 to 72, 96, and 120 HPI, and the mortality rate was 75%, 0%, and 0% for animals inoculated with EHV-9, EHV-1 strain Ab4p, and zebra-borne EHV-1 viruses, respectively. Inoculated hamsters had varying degrees of rhinitis and lymphoplasmacytic meningoencephalitis, as well as differences in the severity and distribution of cerebral lesions. Furthermore, the cellular distribution of viral antigen depended on the inoculated virus. Neuronal necrosis was widely detected in animals inoculated with EHV-9, while marked perivascular cuffs of infiltrating inflammatory cells and gliosis were detected in animals inoculated with EHV-1 strain Ab4p and zebra-borne EHV-1. In the present study, 3 viruses belonging to the herpesvirus family induced encephalitis after initial propagation in the nasal cavity. These viruses might travel to the brain via the olfactory pathway and/or trigeminal nerve, showing different distributions and severities of neuropathological changes.

Neurological alterations induced by formulated imidacloprid toxicity in Japanese quails.

Agrochemical risk assessment that takes into account only pesticide active ingredients without the spray adjuvants will miss important toxicity outcomes detrimental to non-target species including birds. In the present study toxicity of imidacloprid (IMI) pesticide was evaluated individually and in a mixture with polyethylene glycol (PEG-600) as adjuvant against Japanese quails. Oral intubation was used to obtain concentration-mortality data. Oral intubation was used to obtain concentration-mortality data. Treatments of quails for 24h with different doses leading to the calculation of LC50 values. PEG enhances the pesticide efficacy and the LD50 value of IMI was 17.02mg/Kg1, and in combination with PEG it was 15.98mg/kg-1. In the second phase of the study, the effects of a single acute dose of IMI (1/4 LD50) individually or in a mixture with PEG has a potent effect on the activity of plasma AChE and brain monoamines transmitters. However, the addition of PEG-adjuvant to the selected insecticide has shown more toxic potential, more highly significant decreases in AChE activity and different changes in cortical monoamines concentration. In the present study the maximum significant inhibition of AChE activity, was recorded post 72h exposure to IMI individually and 96h in a mixture with PEG and exhibited -37.56% and -32.65% decreases, respectively. Moreover, the oral intubation of IMI individually or in a mixture with PEG caused a significant elevation in the quail cortical NE and 5-HT. The result also showed while the mixture of IMI + PEG induced the more potent effect in DA alterations, IMI individually was more effective in 5-HT changes. Our findings also indicated that PEG exposure induced remarkable changes in the studied monoamines level and the values were significant throughout the tested periods in DA. Moreover, the studied dose level was vigorously affected quail brain cerebral cortex histological structure. When administered individually or in a mixture with PEG, IMI disclosed neural congestion, neuronal degeneration, pyknosis and perivascular cuffing with glial cells.

Analysis of pulmonary vascular injury and repair during Pseudomonas aeruginosa infection-induced pneumonia and acute respiratory distress syndrome.

Herein we describe lung vascular injury and repair using a rodent model of Pseudomonas aeruginosa pneumonia-induced acute respiratory distress syndrome (ARDS) during: 1) the exudative phase (48-hour survivors) and 2) the reparative/fibro-proliferative phase (1-week survivors). Pneumonia was induced by intratracheal instillation of P. aeruginosa strain PA103, and lung morphology and pulmonary vascular function were determined subsequently. Pulmonary vascular function was assessed in mechanically ventilated animals in vivo (air dead space, PaO2, and lung mechanics) and lung permeability was determined in isolated perfused lungs ex vivo (vascular filtration coefficient and extravascular lung water). At 48 hours post infection, histological analyses demonstrated capillary endothelial disruption, diffuse alveolar damage, perivascular cuffs, and neutrophil influx into lung parenchyma. Infected animals displayed clinical hallmarks of ARDS, including increased vascular permeability, increased dead space, impaired gas exchange, and decreased lung compliance. Overall, the animal infection model recapitulated the morphological and functional changes typically observed in lungs from patients during the exudative phase of ARDS. At 1 week post infection, there was lung histological and pulmonary vascular functional evidence of repair when compared with 48 hours post infection; however, some parameters were still impaired when compared with uninfected controls. Importantly, lungs displayed increased fibrosis and cellular hyperplasia reminiscent of lungs from patients during the fibro-proliferative phase of ARDS. Control, sham inoculated animals showed normal lung histology and function. These data represent the first comprehensive assessment of lung pathophysiology during the exudative and reparative/fibro-proliferative phases of P. aeruginosa pneumonia-induced ARDS, and position this pre-clinical model for use in interventional studies aimed at advancing clinical care.

Evaluation of Viral Migration of Different Variants of Equid Alphaherpesvirus 1 in the Central Nervous System of Hamsters by Immunohistochemistry

The intranasal inoculation of equid alphaherpesvirus1 (EHV-1) Brazilian variants A4/72, A9/92, A3/97, Iso72/10 and the Argentine variant AR4 in a Syrian hamster model <i>Mesocricetus auratus</i> induced severe encephalitis. Clinical signs included weight loss, lethargy, somnolence, anorexia, and intense salivation two days post-inoculation (dpi), followed by neurological signs such as loss of proprioception, walking in circles, spastic paralysis, seizures, recumbency and death at 3^rd dpi (A9/92 and A4/72 variants) and 4^th dpi. Respiratory signs such as dyspnea and serosanguinous nasal discharge were also observed. Histopathological changes in brain included mixed inflammatory infiltrate with predominance of mononuclear cells, neuronal degeneration, liquefactive necrosis, hemorrhagic foci, leptomeningitis, perivascular edema, mononuclear infiltration, and perivascular cuffing. Immunohistochemical examination showed viral replication in neurons restrict predominantly to olfactory bulb and frontal cortex (variants AR4 and A3/97) and in groups of cells from distant regions, such as the caudal diencephalon and rostral mesencephalon (variants Iso72/10) and absence of viral antigen labeling of variants A9/92 and A4/72 despite these variants were the most neurovirulent, so new experiments not staggered in days but in hours post inoculation are needed to better understand the viral migration of these variants.

Japanese encephalitis virus-induced neuropathology in mouse model infected through the conjunctival route.

Background & objectives:Mouse is a preferred animal model for studying pathogenesis of Japanese encephalitis virus (JEV) infections, and different routes of inoculation have been tried. Some neurotropic viruses can reach the brain following infection through ocular route. This study was undertaken to establish JEV-induced clinical disease in mouse model through conjunctival route and document the neuropathological effects.Methods:Ten two-week old Swiss albino mice were inoculated with 5 μl Vero cell cultured virus containing 104.7 TCID50 JEV through conjunctival route. Clinical signs of mice were observed twice daily. After necropsy examination, different organs including eyes and olfactory bulbs were collected for histopathological examination, quantification of viral copy number and antigen by real-time TaqMan assay and immunohistochemistry, respectively.Results:Infected mice showed characteristic clinical signs of JE by 4 days post-infection (dpi). Histopathological lesions in brain included perivascular cuffing by mononuclear cells, focal gliosis, necrosis of neurons and neuronophagia and astrocytosis in the cerebrum, cerebellum and the brainstem. JEV viral load was highest in the brain followed by intestine, heart, liver, spleen, lung and kidney. JEV antigen was detected in the bipolar and ganglion cells of the retina and in the mitral cells and periglomerular cells of olfactory bulb and other parts of the brain.Interpretation & conclusions:JEV infection in mice through conjunctival route produced characteristic clinical signs of the disease and neuropathological lesions. Demonstration of JEV antigen in association with neuropathological lesions in the central nervous system and neuronal cells of the eye showed that conjunctival route could be an effective alternate route for virus invasion into the brain. These findings have biosafety implications for researchers, veterinary practitioners and pig farmers.

Pathological and molecular investigation of porcine sapelovirus infection in naturally affected Indian pigs

The aim of the present study was to pathological and molecular investigation of porcine sapelovirus (PSV) in naturally infected Indian pigs of various age groups. Eight samples (16%) out of 49 necropsied animals were positive for PSV on the basis of pathological and molecular investigation. Major lesions of PSV positive cases were thickening and clouding of meninges, congestion in brain, severe to moderate congestion in lungs along with froathy exudates in trachea, thickening of intestinal mucosa, especially mucosal folds of ileum. Microscopic lesions of PSV positive cases in CNS were perivascular cuffing, neuronophagia and focal gliosis. In lungs, interstitial pneumonia was noticed in all cases, and intestinal lesions comprised of sloughing of villi epithelium, moderate to severe congestion of blood vessels and infiltration of mononuclear cells mainly plasma cells in both large and small intestine. RT-PCR results of total cases examined for PSV were targeted for PSV 3D Polymerase, 5′UTR region and VP1 gene respectively. Genetic characterization was done on the basis of viral capsid protein 1 (VP1) gene of PSV. The sequencing and phylogenetic analysis of amplified VP1 gene product showed maximum identity 85–90% with South Korean, KJ821021.1 and Indian, KY053835.1 strain of PSV. Further explorative surveillance and epidemiological studies are suggested to find out the real impact of this economically important disease affecting pigs population of India.

AAV-Mediated Progranulin Delivery to a Mouse Model of Progranulin Deficiency Causes T Cell-Mediated Toxicity.

Adeno-associated virus-mediated gene replacement is emerging as a safe and effective means of correcting single-gene mutationsaffecting the CNS. AAV-mediated progranulin gene (GRN) delivery has been proposed as a treatment for GRN-deficient frontotemporal dementia and neuronal ceroid lipofuscinosis, and recent studies using intraparenchymal AAV-Grn delivery to brain have shown moderate success in histopathologic and behavioral rescue in mouse models. Here, weused AAV9 to deliver GRN to the lateral ventricle to achievewidespread expression in the Grn null mouse brain. We found that, despite a global increase in progranulin, overexpression resulted in dramatic and selective hippocampal toxicityand degeneration affecting neurons and glia. Hippocampal degeneration was preceded by Tcell infiltration and perivascular cuffing. GRN delivery with an ependymal-targeting AAV for selective secretion of progranulin into the cerebrospinal fluid similarly resulted in Tcell infiltration, as well as ependymal hypertrophy. Interestingly, overexpression of GRN in wild-type animals also provoked Tcell infiltration. These results call into question the safety of GRN overexpression in the CNS,with evidence for both a region-selective immune response and cellular proliferative response. Our results highlight the importance of careful consideration of target gene biology and cellular response to overexpression prior to progressing to theclinic.