Emanuele Ricci

Anti-fibrotic effects of fresh and cryopreserved human amniotic membrane in a rat liver fibrosis model

The human amniotic membrane (hAM), thanks to its favorable properties, including anti-inflammatory, anti-fibrotic and pro-regenerative effects, is a well-known surgical material for many clinical applications, when used both freshly after isolation and after preservation. We have shown previously that hAM patching is a potential approach to counteract liver fibrosis. Indeed, when fresh hAM was used to cover the liver surface of rats with liver fibrosis induced by the bile duct ligation (BDL) procedure, the progression and severity of fibrosis were significantly reduced. Since cryopreservation enables safety and long-term storage of hAM but may influence its functional properties, here we compared the anti-fibrotic effects of fresh and cryopreserved hAM in rats with BDL-induced liver fibrosis. After BDL, the rat liver was covered with a piece of fresh or cryopreserved hAM, or left untreated. Six weeks later, the degree of liver fibrosis was assessed histologically using the Knodell and the METAVIR scoring systems. Digital image analysis was used to quantify the percentage of the areas of each liver section displaying ductular reaction, extracellular matrix (ECM) deposition, activated myofibroblasts and hepatic stellate cells (HSCs). Liver collagen content was also determined by spectrophotometric technique. The degree of liver fibrosis, ductular reaction, ECM deposition, and the number of activated myofibroblasts and HSCs were all significantly reduced in hAM-treated rats compared to control animals. Fresh and cryopreserved hAM produced the same anti-fibrotic effects. These findings indicate that cryopreservation maintains the anti-fibrotic properties of hAM when used as a patch to reduce the severity of liver fibrosis.

Molecular isoforms of high-mobility group box 1 are mechanistic biomarkers for epilepsy

Approximately 30% of epilepsy patients do not respond to antiepileptic drugs, representing an unmet medical need. There is evidence that neuroinflammation plays a pathogenic role in drug-resistant epilepsy. The high-mobility group box 1 (HMGB1)/TLR4 axis is a key initiator of neuroinflammation following epileptogenic injuries, and its activation contributes to seizure generation in animal models. However, further work is required to understand the role of HMGB1 and its isoforms in epileptogenesis and drug resistance. Using a combination of animal models and sera from clinically well-characterized patients, we have demonstrated that there are dynamic changes in HMGB1 isoforms in the brain and blood of animals undergoing epileptogenesis. The pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset and prospectively identified animals that developed the disease. Consistent with animal data, we observed early expression of disulfide HMGB1 in patients with newly diagnosed epilepsy, and its persistence was associated with subsequent seizures. In contrast with patients with well-controlled epilepsy, patients with chronic, drug-refractory epilepsy persistently expressed the acetylated, disulfide HMGB1 isoforms. Moreover, treatment of animals with antiinflammatory drugs during epileptogenesis prevented both disease progression and blood increase in HMGB1 isoforms. Our data suggest that HMGB1 isoforms are mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy in humans, necessitating evaluation in larger-scale prospective studies.

Cannabinoid receptor type 1 and 2 expression in the skin of healthy dogs and dogs with atopic dermatitis

OBJECTIVE To determine the distribution of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) in skin (including hair follicles and sweat and sebaceous glands) of clinically normal dogs and dogs with atopic dermatitis (AD) and to compare results with those for positive control samples for CB1 (hippocampus) and CB2 (lymph nodes). SAMPLE Skin samples from 5 healthy dogs and 5 dogs with AD and popliteal lymph node and hippocampus samples from 5 cadavers of dogs. PROCEDURES CB1 and CB2 were immunohistochemically localized in formalin-fixed, paraffin-embedded sections of tissue samples. RESULTS In skin samples of healthy dogs, CB1 and CB2 immunoreactivity was detected in various types of cells in the epidermis and in cells in the dermis, including perivascular cells with mast cell morphology, fibroblasts, and endothelial cells. In skin samples of dogs with AD, CB1 and CB2 immunoreactivity was stronger than it was in skin samples of healthy dogs. In positive control tissue samples, CB1 immunoreactivity was detected in all areas of the hippocampus, and CB2 immunoreactivity was detected in B-cell zones of lymphoid follicles. CONCLUSIONS AND CLINICAL RELEVANCE The endocannabinoid system and cannabimimetic compounds protect against effects of allergic inflammatory disorders in various species of mammals. Results of the present study contributed to knowledge of the endocannabinoid system and indicated this system may be a target for treatment of immune-mediated and inflammatory disorders such as allergic skin diseases in dogs.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 hours, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared to wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared to vehicle control. In light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.

Blunt Force Trauma in Veterinary Forensic Pathology

Veterinary pathologists commonly encounter lesions of blunt trauma. The development of lesions is affected by the object’s mass, velocity, size, shape, and angle of impact and by the plasticity and mobility of the impacted organ. Scrape, impact, and pattern abrasions cause localized epidermal loss and sometimes broken hairs and implanted foreign material. Contusions are best identified after reflecting the skin, and must be differentiated from coagulopathies and livor mortis. Lacerations—traumatic tissue tears—may have irregular margins, bridging by more resilient tissue, deviation of the wound tail, crushed hairs, and unilateral abrasion. Hanging or choking can cause circumferential cervical abrasions, contusions and rupture of hairs, hyoid bone fractures, and congestion of the head. Other special forms of blunt trauma include fractured nails, pressure sores, and dog bites. Ocular blunt trauma causes extraocular and intraocular hemorrhages, proptosis, or retinal detachment. The thoracic viscera are relatively protected from blunt trauma but may develop hemorrhages in intercostal muscles, rib fractures, pulmonary or cardiac contusions or lacerations with subsequent hemothorax, pneumothorax, or cardiac arrhythmia. The abdominal wall is resilient and moveable, yet the liver and spleen are susceptible to traumatic laceration or rupture. Whereas extravasation of blood can occur after death, evidence of vital injury includes leukocyte infiltration, erythrophagocytosis, hemosiderin, reparative lesions of fibroblast proliferation, myocyte regeneration in muscle, and callus formation in bone. Understanding these processes aids in the diagnosis of blunt force trauma including estimation of the age of resulting injuries.

Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: Effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation

It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrP(d)) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14-17 days post-inoculation [dpi]) or late (175-201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrP(d) in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrP(d) in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrP(d) negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrP(d) positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrP(d) accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrP(d) accumulation. An additional observation of the study was that sheep that were methionin-threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrP(d) accumulation in the LRS and brain and increased survival times, suggesting an influence of such polymorphism in the susceptibility to scrapie.

Fibrocartilaginous Embolic Myelopathy in a Lion (Panthera leo)

Abstract A 6-yr-old, captive-born male lion (Panthera leo) with a 3-day history of acute and nonprogressive spastic paraplegia of the hind limbs and flaccid paraplegia of the left forelimb, was submitted for postmortem examination. Before and after the onset of the neurologic signs, neither hematologic nor other significant clinical abnormalities were observed. The only remarkable gross lesion was restricted to the C6–C7 segments of the spinal cord, where a focal and asymmetric enlargement of the cord displaced the nerve roots. On the cut surface, a poorly demarcated dark red hemorrhagic lesion involved the lateroventral funiculi and the ventral horn of the gray matter, exclusively on the left half of the cord. Extensive necrosis, hemorrhages, degeneration of the neuroparenchyma, and several fibrocartilaginous emboli occluding the lumina of intraparenchymal arteries were present within histologic sections of spinal cord. Emboli also were detected within the meningeal vessels. This is the first report of fibrocartilaginous embolic myelopathy occurring in a lion.

MRI findings, surgical treatment and follow-up of a myelomeningocele with tethered spinal cord syndrome in a cat:

A 7-month-old male neutered cat was referred for paraparesis and painful sensation at the level of T13 vertebra where a dermal cyst was observed. Spine radiographs and magnetic resonance imaging (MRI) showed a well-encapsulated cyst communicating with the meninges and spinal cord, suggestive of hydromyelia and myelodysplasia. Dorsal laminectomy was performed and the cyst was completely removed. The day after surgery, the cat was ambulatory paraparetic. Involuntary defecation was observed for only a few days. The surgical specimen was cystic and covered by skin. Microscopic examination revealed a hollow hemispheric mass of glial fibrillary acidic protein (GFAP)-positive neural tissue lined by ependyma and formed of glia and vascular structures consistent with myelomeningocele (MMC). Only anecdotal descriptions of MMC have been published in the veterinary literature, mainly in the lumbosacral spinal cord. To the authors’ knowledge, this is the first report of a MMC with tethered spinal cord syndrome in a cat successfully treated surgically.

High mobility group box 1 in the inflammatory pathogenesis of epilepsy: profiling circulating levels after experimental and clinical seizures

Abstract Background High mobility group box 1 (HMGB1) is a chromatin binding protein that is passively released by necrotic cells and actively secreted in response to inflammatory stimuli in a hyperacetylated form. HMGB1 is upregulated in the brain after injury and can regulate localised inflammatory reactions resulting in seizures. Antiepileptic drugs prevent seizures but have no disease modifying effect or influence on natural history. Immunomodulatory antiepileptic drugs have huge potential, but can have serious adverse effects. Patient stratification is required to maximise the benefit to risk ratio. We investigated serum and brain concentrations of HMGB1 in rodent models of seizures and epilepsy and in people with refractory epilepsy. Methods We used three experimental models in this study. Adult male C57BL/6J mice received repeated intraperitoneal injections of kainic acid (KA) until the onset of convulsive status epilepticus; status epilepticus was terminated after 2 h with diazepam, with brain and blood samples obtained at 3, 6, 24, and 72 h and 7 and 14 days thereafter. Tonic seizures were induced in adult male CF1 mice by maximal electroshock (MES) delivered via corneal electrodes, with brain and blood samples obtained at 1, 4, 8, 16, and 24 h after seizure induction. Finally, serial samples were obtained at baseline, 1, 4, 8, and 12 h after an observed seizure from people with drug refractory epilepsy undergoing videoelectroencephalograph telemetry. Total HMGB1 expression was measured by western blot and immunohistochemistry in brain and by ELISA in serum. Expression of individual HMGB1 isoforms was determined by liquid-chromatography tandem mass-spectrometry (LC-MS/MS). Findings HMGB1 expression was significantly raised in hippocampus and cortex (p vs 3 h 16·5 [1·5], p vs 4 h 19·7 [11·3], p vs 4 h 15·2 [7·0], p vs 0·7 [0·3], p vs 1·25 [0·71], p Interpretation These findings suggest that blood and brain HMGB1 concentrations are increased as a result of seizures in both animal models and patients with epilepsy. Increases in HMGB1 might occur as a consequence of seizures. Patients with refractory epilepsy have higher baseline HMGB1, which might predispose them to recurrent seizure activity. The association between HMGB1 and seizures requires further exploration. Funding UK Medical Research Council, ICON, GlaxoSmithKline, AstraZeneca, the Medical Evaluation Unit.

Encephalitozoon cuniculi in rabbits: Serological screening and histopathological findings

Serological prevalence of E. cuniculi infection was assessed in 183 rabbits from central Italy. In seropositive deceased rabbits, histopathological lesions were also evaluated. Sera from 118 rabbits from 6 intensive farms, 10 rabbits from 6 family farms, 16 rabbits from a zoo, 30 rabbits from 5 research laboratories and 9 pet rabbits from 9 different owners, were tested by an enzyme-linked immunosorbent assay. Data were statistically analysed. Tissue samples from brain and kidney of 10 deceased rabbits were formalin-fixed and subsequently analysed by histopathology and immunohistochemistry. Anti-E. cuniculi antibodies were found in 129/183 (70.5%) analysed sera. At statistical analysis, E. cuniculi seropositivity was significantly higher (p<0.05) in industrial and zoo rabbits. At histology, different degrees of pathological lesions were found in serological positive (9) deceased animals. In three rabbits deceased after showing neurological signs, the severity of the lesions was interpreted as a likely cause for their death.