Adeno-associated virus serotype 9 (AAV9)-mediated gene transfer has been reported in central nervous system (CNS) and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP) across the mouse CNS and selected peripheral tissues after intrathecal (i.t.) or intravenous (i.v.) delivery of equivalent doses of single-stranded AAV9 vector. After i.t. delivery, GFP immunoreactivity (-ir) was observed in spinal neurons, primary afferent fibers and corresponding primary sensory neurons at all spinal levels. Robust transduction was seen in small and large dorsal root ganglion (DRG) neurons as well as trigeminal and vagal primary afferent neurons. Transduction efficiency in sensory ganglia was substantially lower in i.v. treated mice. In brain, i.v. delivery yielded GFP-immunoreactivity (-ir) primarily in spinal trigeminal tract, pituitary, and scattered isolated neurons and astrocytes. In contrast, after i.t. delivery, GFP-ir was widespread throughout CNS, with greater intensity and more abundant neuropil-like staining at 6 weeks compared to 3 weeks. Brain regions with prominent GFP-ir included cranial nerve nuclei, ventral pons, cerebellar cortex, hippocampus, pituitary, choroid plexus, and selected nuclei of midbrain, thalamus and hypothalamus. In cortex, GFP-ir was associated with blood vessels, and was seen in both neurons and astrocytes. In the periphery, GFP-ir in colon and ileum was present in the enteric nervous system in both i.v. and i.t. treated mice. Liver and adrenal cortex, but not adrenal medulla, also showed abundant GFP-ir after both routes of delivery. In summary, i.t. delivery yielded higher transduction efficiency in sensory neurons and the CNS. The observation of comparable gene transfer to peripheral tissues using the two routes indicates that a component of i.t. delivered vector is redistributed from the subarachnoid space to the systemic circulation.

VGF (non-acronymic) is a granin-like protein that is packaged and proteolytically processed within the regulated secretory pathway. VGF and peptides derived from its processing have been implicated in neuroplasticity associated with learning, memory, depression, and chronic pain. In sensory neurons, VGF is rapidly increased following peripheral nerve injury and inflammation. Several bioactive peptides generated from the C-terminus of VGF have pro-nociceptive spinal effects. The goal of the present study was to examine the spinal effects of the peptide TLQP-21 and determine whether it participates in spinal mechanisms of persistent pain. Application of exogenous TLQP-21 induced dose-dependent thermal hyperalgesia in the warm water immersion tail withdrawal test. This hyperalgesia was inhibited by a p38 MAPK inhibitor as well as inhibitors of cyclooxygenase and lipoxygenase. We used immunoneutralization of TLQP-21 to determine the function of the endogenous peptide in mechanisms underlying persistent pain. In mice injected intradermally with complete Freund’s adjuvant, intrathecal treatment with anti-TLQP21 IgG immediately prior to or 5 h after induction of inflammation dose-dependently inhibited tactile hypersensitivity and thermal hyperalgesia. Intrathecal anti-TL21 administration also attenuated the development and maintenance of tactile hypersensitivity in the spared nerve injury model of neuropathic pain. These results provide evidence that endogenous TLQP-21 peptide contributes to the mechanisms of spinal neuroplasticity after inflammation and nerve injury.

Background Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno-associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV-driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. Methods Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. GFP labeling in DRG and colon was examined using immunohistochemistry. Key Results Analysis of colon from rAAV8-GFP treated mice demonstrated GFP-immunoreactivity (GFP-ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa and mucosa, but not in cell bodies of enteric neurons. Notably, GFP-ir co-localized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular-like clusters surrounding nuclei within myenteric ganglia. Additionally, GFP-positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP-ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. Conclusions and Inferences These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.

Lymphoma is a common hematopoietic neoplasm of dogs. A definitive diagnosis typically requires the collection of samples via fine‐needle aspirate or biopsy. A unique case of canine renal T‐cell lymphoma diagnosed using urine sediment microscopy with flow cytometry and PCR for Antigen Receptor Rearrangement (PARR) is presented. A fresh urine sample was collected via a urinary catheter and immediately prepared for cytologic examination, flow cytometry, and PARR. The flow cytometric study revealed that 83% of the cells were large CD3+CD8+ T cells, while PARR identified a clonally rearranged T‐cell receptor gene, supporting the flow cytometry findings. Despite supportive care, the patient progressed to anuric renal failure and was humanely euthanized. A necropsy was performed, and tissues from the upper and lower urinary tracts were collected. Histologically, the right and left kidneys were infiltrated by a neoplastic round cell population effacing the cortex and medulla. Immunohistochemistry for the T‐ and B‐cell antigens CD3 and CD20, respectively, revealed that the neoplastic population within the kidney demonstrated diffuse, strong, membranous to intracytoplasmic CD3 expression while lacking CD20 expression. These results confirmed the diagnosis of renal T‐cell lymphoma. This is the first known report of canine lymphoma diagnosed using either urine flow cytometry or clonality testing. Therefore, in select cases, urine flow cytometry and/or PARR are feasible to perform on urine‐derived cells as a quick and cost‐effective means to aid in the diagnosis of urinary tract lymphoma.

A free-ranging, female American black bear (Ursus americanus) was observed and radio-tracked by researchers from the Wildlife Research Institute near Ely (St. Louis County), Minnesota, starting in June of 2004. Body weight was periodically recorded at a field weight station and demonstrated typical seasonal pre-hibernation weight gain, with recordings varying from 70 to 182 kg. The last weight obtained at the field station in August of 2009 was 102 kg, which was considered normal for the time of year. However in late November, the bear was observed to be severely underweight and inappetent. Shortly thereafter, the bear was found deceased, partially submerged in a shallow pool of water. The carcass was recovered and submitted to the University of Minnesota Veterinary Diagnostic Laboratory (St. Paul, Minnesota) for diagnostic evaluation.At presentation, the bear weighed 62 kg. The haircoat of the trunk, rump, and limbs was thinned and unkempt, and scattered within the denser haircoat of the brisket were numerous round, dorsoventrally flattened, white to brown, small (1-3 mm diameter) lice, identified as Trichodectes pinguis euarctidos.Necropsy confirmed generalized marked skeletal muscle atrophy and visible but inadequate subcutaneous and visceral adipose stores. Bilaterally, the lungs were diffusely voluminous and discolored pale gray to tan (Fig. 1). On cut section, the pulmonary parenchyma was meaty, soft, and mottled tan to gray with a small amount of brown mucoid material within the larger airways (Fig. 2). The tracheobronchial, mesenteric, perirenal, iliac, inguinal, and colonic lymph nodes were enlarged up to approximately 3 times normal size and, on section, were multifocally to diffusely effaced and expanded by homogenous, tan-yellow, soft tissue with occasional white, firm foci. Other lesions included infrequent strands of fibrin within the peritoneal cavity, mild diffuse hepatomegaly, focal chronic ulceration of the proximal Abstract. An aged, free-ranging, female, radio-collared American black bear (Ursus americanus) died after an approximately 5 month long period of weight loss. Gross necropsy findings included severe diffuse pyogranulomatous bronchopneumonia, marked granulomatous lymphadenitis of tracheobronchial lymph nodes and multiple intra-abdominal lymph nodes, chronic focal jejunal ulceration, and widespread alopecia. Histopathologic examination revealed abundant fungal organisms morphologically compatible with Blastomyces sp. within pyogranulomatous inflammatory lesions in the lungs, multiple lymph nodes, liver, kidneys, jejunum, and right adrenal gland. In addition, the haircoat had a mild infestation of chewing lice (Trichodectes pinguis euarctidos), and large numbers of rhabditid nematodes consistent with Pelodera sp. were histologically observed within hair follicles.