Expression Of Protein Gene Product Research Articles

Human Placenta-Derived Mesenchymal Stem Cells Ameliorate Diabetic Neuropathy via Wnt Signaling Pathway.

To investigate the effect of placenta-derived mesenchymal stem cells (PMSCs) on diabetic peripheral neuropathy and explore the role of Wnt signaling pathway. Twenty-seven male db/db mice were randomly categorized into the control group, PMSC group, and PMSC treatment with Wnt inhibitor treatment group. Intervention was initiated in week 22. Thermal stimulation response was determined with a plantar analgesia tester. The mice were sacrificed on 7, 14, and 28 days. The morphology of sciatic nerves was observed by electron microscopy, and the expression of protein gene product (PGP) 9.5, S100β, and Ku80 was detected by immunofluorescence. Bax, β-catenin, and dishevelled1 (DVL1) were detected by western blot. Thermal stimulation response was improved in the PMSC group on 14 and 28 days. Compared with the control group, PGP9.5 was increased in the PMSC group, accompanied by a significant increase in the expression of S100β. On the contrary, LGK974 inhibited the effect of PMSCs on thermal stimulation response and the expression of PGP9.5 and S100β. Both PGP9.5 and S100β were correlated with Ku80 in fluorescence colocalization. The myelin sheath of sciatic nerves in the PMSC group was uniform and dense compared with that in the control group. The effects of PMSCs promoting myelin repair were significantly inhibited in the PMSC+LGK974 group. Bax in the PMSC group expressed less than the control group. In contrast, the expressions of β-catenin and DVL1 were higher compared with that in the control group on the 14th and 28th days. The expression of DVL1 and β-catenin was lower in the PMSC+LGK974 group than in the PMSC group. PMSCs improved the symptoms of diabetic peripheral neuropathy, along with the improvement of nerve myelin lesions, promotion of nerve regeneration, and activation of Schwann cells, which might be related to the regulation of Wnt signaling pathway and inhibition of apoptosis.

Electroacupuncture at ST36 Improve the Gastric Motility by Affecting Neurotransmitters in the Enteric Nervous System in Type 2 Diabetic Rats.

Electroacupuncture (EA) can effectively relieve hyperglycemia and gastric emptying disorders in diabetic gastroparesis (DGP). However, the effect of EA on type 2 diabetes mellitus (T2DM) gastroparesis and its mechanism in the enteric nervous system (ENS) are rarely studied. We investigated the therapeutic effect of EA at ST36 and its effect on the main inhibitory and excitatory neurotransmitters in the ENS in DGP rats. Male Sprague-Dawley (SD) rats were fed a high-fat diet for 2 weeks and injected with streptozotocin (STZ) at 35 mg/kg to induce T2DM. T2DM rats were divided into the diabetic mellitus (DM) group and the EA group. The control (CON) group comprised normal rats without any intervention. EA treatment was started 6 weeks after the induction of DM and continued for 5 weeks. The body weight and food intake of the rats were recorded every week. Blood glucose, insulin, glucose tolerance, gastric emptying, and antral motility were measured after treatment. The expression of protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT) in gastric antrum were quantified by western blotting and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The T2DM gastroparesis model was successfully established. EA treatment reduced the body weight, food intake, and blood glucose; improved glucose intolerance and insulin resistance; increased the gastric emptying rate, the mean antral pressure, and the amplitude of antral motility; and decreased the frequency of antral motility compared with those in the DM group. EA treatment increased the expression level of nNOS, ChAT, and PGP9.5 proteins, and nNOS and ChAT mRNA. The results suggested that EA at ST36 could ameliorate DGP, partly restore the damage to general neurons, and increase nNOS and ChAT in the gastric antrum. EA improved DGP partly via reducing the loss of inhibitory and excitatory neurotransmitters in the ENS.

Functional and molecular characterisation of the bilateral pelvic nerve crush injury rat model for neurogenic detrusor underactivity.

To create a rat model for neurogenic detrusor underactivity (DU) by bilateral pelvic nerve crush injury (BPNI) and to study temporal changes in detrusor contractility and morphology. Male Sprague-Dawley rats were subjected to BPNI or sham surgery and evaluated at 1, 3 and 9weeks after surgery. Bladder function was determined invivo by awake cystometry, micturition pattern analysis, and 24-h urine collection. Bladders were harvested for invitro pharmacological investigation by isometric tension recording. Bladders and major pelvic ganglia were investigated by quantitative reverse transcription-polymerasechain reaction and histochemistry. Overflow incontinence was observed at 1week after BPNI. At 3 and 9weeks after BPNI, rats showed a bladder phenotype characteristic for DU with increased post-void residual urine volumes, reduced voiding efficiencies, and lower maximum pressures. In isolated bladder strips, contractile responses to KCl, carbachol, and α,β-methylene adenosine 5'-triphosphate (α,β-mATP) were preserved. On the other hand, neural-induced contractility was reduced after BPNI, in line with reduced expression of protein gene product 9.5 and choline acetyltransferase in the major pelvic ganglion at 1week after BPNI. The bladder-to-body weight ratio and detrusor thickness increased after BPNI, indicating detrusor hypertrophy to compensate for the reduced neural input. BPNI induces a rat model for neurogenic DU. In this model, the detrusor maintains its contractility but denervation of the detrusor was observed.

PKCβII-induced upregulation of PGP9.5 and VEGF in postoperative persistent pain in rats.

PurposePostoperative pain is a common clinical problem. In this study, we aimed to investigate the role of protein kinase C βII (PKCβII) in the progression of postoperative pain following skin/muscle incision and retraction (SMIR) surgery.Materials and methodsSMIR postoperative pain model was established in rats, akin to a clinical procedure. The expression level and location of p-PKCβII were observed in dorsal root ganglion (DRG) or spinal cord from SMIR-operated rats by Western blotting and immunofluorescence. In addition, the effects of PKCβII on the expression of protein gene product 9.5 (PGP9.5) or vascular endothelial growth factor (VEGF) were assessed by using pharmacological activator and inhibitor of PKCβII. Moreover, mechanical withdrawal threshold (MWT) was assessed before or after SMIR-operated rats were treated with inhibitor or activator of PKCβII.ResultsThe expression of PKCβII in DRG and spinal cord was significantly increased after SMIR surgery (P < 0.001, P < 0.01) and expression of PKCβII was located in the neurons of the spinal cord, and magnocellular neurons, non-peptide neurons, and peptide neurons in DRG. Besides, compared with skin/muscle incision group, retraction caused a marked increase in the expression of PKCβII and a significant decrease of MWT (P < 0.001, P < 0.05). The activator of PKCβII greatly increased the expression of PGP9.5 and VEGF (P < 0.05, P < 0.01) and enhanced MWT (P < 0.001), while inhibitor of PKCβII decreased the expression of PGP9.5 and VEGF and attenuated MWT (P < 0.05, P < 0.01, P < 0.001).ConclusionActivation of PKCβII signaling pathways might be an important mechanism in the progression of postoperative pain.

Electro‐acupuncture at ST37 and ST25 induce different effects on colonic motility via the enteric nervous system by affecting excitatory and inhibitory neurons

On the basis of the importance of the enteric nervous system (ENS) in gastrointestinal motility, we hypothesized that the ENS may mediate the therapeutic efficacy of electro-acupuncture (EA) in constipation by regulating the mechanisms underlying the effects of EA on gastrointestinal function. Model mice with constipation were generated by gastric instillation of 0-4°C normal saline. Defecation time and stool (form and wet and dry weight) were assessed. The effect of EA at ST37 or ST25 on colorectal motility and proximal colonic motility was assessed using a water-filled balloon. The expression of protein gene product 9.5 (PGP9.5), the cholinergic neuron marker acetyltransferase (ChAT) and the anticholinergic neuron marker nitric oxide synthase (nNOS) was detected by immunohistochemistry, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis. ST37 and ST25 improved colorectal pressure; however, ST37 but not ST25 improved proximal colonic pressure. In the proximal colon, the expression of PGP9.5 returned to normal after EA at ST 37, while EA at ST25 did not have this effect. In addition, qPCR and western blot analysis showed that ST37 could downregulate the expression of nNOS and upregulate the expression of ChAT to normal levels, while ST25 could only downregulate the expression of nNOS to normal levels. Electro-acupuncture at specific acupoints can improve intestinal motility in constipation by altering the ENS and differentially affecting excitatory and inhibitory neurons, restoring the coordination between contraction and relaxation muscles, and working in concert with the central nervous system and peripheral neural pathways.

Characterization of male germ cell markers in canine testis

Spermatogenesis begins at puberty and continues throughout a male’s life. This process is initiated and maintained by spermatogonial stem cells in the seminiferous tubules, and these cells produce haploid spermatozoa. Markers of male germ cells have been fully identified in rodents, including mice and rats, but not in canines. To characterize the canine male germ cells, histological and immunohistochemical analyses were performed, using prepubertal (1–3-month-old), early pubertal (4-month-old), and postpubertal (7-month-old) dog testes. Expression of protein gene product 9.5 (PGP9.5), deleted in azoospermia-like (DAZL), synaptonemal complex protein (SCP3), tyrosine-protein kinase Kit (C-kit), and acrosin was confirmed by immunohistochemical analysis. PGP9.5 and DAZL were detected in spermatogonia and co-localized near the basement membrane of seminiferous tubules. Some SCP3-positive cells expressed PGP9.5 but not C-kit, and most of these cells were located near the basement membrane. C-kit is a marker of differentiated spermatogenic cells. In addition, acrosin was detected in C-kit-positive spematocytes and mature spermatozoa, whereas C-kit was detected in Sertoli cells in all stages of canine testis development. We suggest that male germ cell markers detected in other species are conserved in canines. PGP9.5, DAZL, SCP3, and acrosin expressions were conserved among various species, but C-kit expression varied. This study might facilitate the identification of stage-specific canine germ cell markers and cellular mechanisms of spermatogenesis.

Protein Gene Product 9.5 Expression in Stallion Testes

The expression of protein gene product 9.5 (PGP9.5) in testes has been reported in domestic species, including pigs, sheep, and goats. The main objectives of this study were to: (1) investigate the expression pattern of PGP9.5 in stallion testes and (2) identify stage-dependent immunolabeling of PGP9.5 in stallion testes. Samples were categorized based on the age of the horses as follows: prepubertal (<1 year), pubertal (1–1.5 years), postpubertal (2–3 years), and adult (4–8 years). Immunohistochemistry was performed to investigate the expression pattern of PGP9.5 at different reproductive stages. For double staining, the goat anti-human DAZL and GATA4 antibody were used as markers for germ cells and Sertoli cells, respectively. The Western blot procedure was used to determine the cross-reactivity of the PGP9.5 antibody in horse testis tissues. During the prepubertal and pubertal stages, germ cells, which were located in the center of the seminiferous tubules, were positive for PGP9.5. During the postpubertal and adult stages, expression of PGP9.5 was found in the cytoplasm of germ cells adjacent to the basement membrane. PGP9.5-positive cells were immunolabeled with DAZL but not immunolabeled in Sertoli cells at all reproductive stages. Interestingly, Leydig cells were stained with PGP9.5 at all reproductive stages. In conclusion, PGP9.5 antibody can be used as a tool to identify and isolate PGP9.5-positive germ cells only after Leydig cells were separated from the seminiferous tubules.

Impairments of the primary afferent nerves in a rat model of diabetic visceral hyposensitivity.

BackgroundDiabetic neuropathy in visceral organs such as the gastrointestinal (GI) tract is still poorly understood, despite that GI symptoms are among the most common diabetic complications. The present study was designed to explore the changes in visceral sensitivity and the underlying functional and morphological deficits of the sensory nerves in short-term diabetic rats. Here, we compared the colorectal distension (CRD)-induced visceromotor response (VMR, an index of visceral pain) in vivo, the mechanosensitivity of colonic afferents ex vivo as well as the expression of protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP) in colon between diabetic (3–6 weeks after streptozotocin injection) and control (age-matched vehicle injection) rats.ResultsVMR was markedly decreased in the diabetic compared to the control rats. There was a significant decrease in multiunit pelvic afferent nerve responses to ramp distension of the ex vivo colon and single unit analysis indicated that an impaired mechanosensitivity of low-threshold and wide dynamic range fibers may underlie the afferent hyposensitivity in the diabetic colon. Fewer PGP 9.5- or CGRP-immunoreactive fibers and lower protein level of PGP 9.5 were found in the colon of diabetic rats.ConclusionsThese observations revealed the distinctive feature of colonic neuropathy in short-term diabetic rats that is characterized by a diminished sensory innervation and a blunted mechanosensitivity of the remnant sensory nerves.

Role of the skin expression of neuropeptides, neurotrophins and their receptors in the pathogenesis of dermatoses

to define the role of neurotransmitters and their receptors in the development of itch and in the maintenance of a skin inflammatory response in patients with psoriasis and atopic dermatitis. Skin biopsy specimens from 30 patients with psoriasis and 30 patients with atopic dermatitis were investigated by histological, immunoperoxidase, and indirect immunofluorescence assays. The investigators determined the expression of protein gene product 9.5 (PGP9.5), amphiregulin, semaphorin 3A, calcitonin gene-related peptide (CGRP) and its receptor (CGRP-R), nerve growth factor (NGF) and its receptor TrkA, and substance P (SP) and its receptor SP-R. The indirect immunofluorescence assay was used for quantitative analysis. The findings were statistically analyzed using a Statistica 10 program. Immunoperoxidase examination of the skin biopsy specimens from patients with atopic dermatitis and psoriasis revealed enhanced expression of amphiregulin, NGF, and PGP9.5, appearance of positively stained epidermal nerve fibers, and decreased expression of the nerve reduction factor semaphorin 3A in all cases. Some patients with atopic dermatitis and psoriasis showed increased expression of CGRP and CGRP-R, SP, SP-R, and TrkA. A pronounced inflammatory response was generally observed in these cases. The investigation performed suggests that atopic dermatitis and psoriasis are characterized by a larger number of epidermal nerve fibers and by a direct correlation between this indicator, disease severity, and itch intensity. The production of neuropeptides and neurotrophins is closely related to the development of a skin inflammatory response irrespective of its cause and dysregulation of these processes is likely to favor the body's sensitization and the chronic pattern the course of diseases.

The clinicopathological significance of neurogenesis in breast cancer.

BackgroundRecent reports support a novel biological phenomenon about cancer related neurogenesis. However, little is known about the clinicopathological significance of neurogenesis in breast cancer.MethodsA total of 196 cases, including 20 of normal tissue, 14 of fibroadenoma, 18 of ductal carcinoma in situ (DCIS) and 144 of invasive ductal carcinoma (IDC) of the breast were used. The tissue slides were immunostained for protein gene product (PGP) 9.5 and S 100 to identify nerves. The correlation between the expression of PGP 9.5 and clinicopathological characteristics in IDC of the breast was assessed.ResultsWhile the PGP 9.5 positive nerve fibers are identified in all cases of normal breast tissue controls and in the tumor stroma of 61% (89/144) cases of invasive ductal carcinomas, PGP 9.5 positive nerve fibers are not seen in the tumor stroma of cases of fibroadenoma and DCIS. The percentage of tumors that exhibited neurogenesis increased from tumor grade I to tumor grade II and III (29.4% vs 71.8%, p < 0.0001). In addition, patients with less than 3 years of disease-free survival tended to have a higher positive expression of PGP 9.5 compared to patients with an equal or more than 3 years of disease-free survival (64.8% vs 46.7%, p = 0.035). Furthermore, moderate/strong expression of PGP 9.5 was found to be significantly related to microvessel density (MVD, p = 0.014). Interestingly, PGP 9.5 expression was significantly associated with higher MVD in the ER-negative (p = 0.045) and node-negative (p = 0.039) subgroups of IDC of the breast.ConclusionsThis data indicates that neurogenesis is associated with some aggressive features of IDC including tumor grade and patient survival as well as angiogenesis, especially in ER-negative and node-negative subtypes of IDC of the breast. Thus, neurogenesis appears to be associated with breast cancer progression and may play a role in therapeutic guidance for patients with ER-negative and node-negative invasive breast cancer.

Expression of Protein Gene Product 9.5 and Sal-like Protein 4 in Canine Seminomas

The aim of the present study was to characterize canine classical seminoma (SE) and spermatocytic seminoma (SS) by immunohistochemical expression of gonocytic and spermatogonial cellular markers (c-Kit, placental alkaline phosphatase [PLAP], protein gene product 9.5 [PGP9.5]and Sal-like protein 4 [Sall4]) and histochemically by the periodic acid-Schiff (PAS) reaction. Twenty-five cases of SE and 23 cases of SS were investigated. Two cases of dysgerminoma were also examined. c-Kit was expressed on the cell membrane of 13 of 25 cases of SE (52%) and four of 23 cases of SS (16%). This marker was not expressed in dysgerminoma. PLAP immunoreactivity was observed in the cytoplasm of neoplastic cells of six of 25 cases of SE (24%). PLAP was not expressed in cases of SS and dysgerminoma. All samples of SE, SS and dysgerminoma showed cytoplasmic expression of PGP9.5 and nuclear immunoreactivity for Sall4. There was fine granular cytoplasmic PAS staining in neoplastic cells in five of 25 cases of SE (20%), while all samples of SS and dysgerminoma cases were PAS negative. These findings suggest that it is not possible to differentiate canine SE and SS using these markers. This may be because canine SS may be derived from spermatogonia that can differentiate to spermatocytesand also because cases of canine SE might consist of neoplastic cells that have lost their gonocytic nature. This study was the first to show positive immunoreactivity for Sall4 in canine seminomas and dysgerminomas and expression of PGP9.5 in canine dysgerminomas.

Immunohistochemical Expression of Melanocytic Antigen PNL2, Melan A, S100, and PGP 9.5 in Equine Melanocytic Neoplasms

The immunoreactivity of PNL2, Melan A, and protein gene product (PGP) 9.5 was compared with that of S100 protein in 50 formalin-fixed, paraffin-embedded equine melanocytic neoplasms. PNL2, PGP 9.5, and S100 protein were detected in all 50 neoplasms; none expressed Melan A. PNL2 was not expressed in 62 nonmelanocytic tumors (equine sarcoids, schwannomas, carcinomas, sarcomas, endocrine tumors, sex-cord stromal tumors, germ cell tumors, and leukocytic tumors) or in normal tissues other than epidermis. In summary, antibody PNL2 is a sensitive marker of equine melanocytic neoplasms and is more specific than S100 protein or PGP 9.5. In contrast, the monoclonal antibody to Melan A did not react with any of the equine melanomas.

Relationship between neural alteration and perineural invasion in pancreatic cancer patients with hyperglycemia.

BackgroundPatients with higher levels of fasting serum glucose have higher death rates from pancreatic cancer compared to patients with lower levels of fasting serum glucose. However, the reasons have not been studied. The goal of the current study was to examine the neural alterations in pancreatic cancer patients with hyperglycemia and to identify the relationship between the neural alterations and perineural invasion.Methodology/Principal FindingsThe clinical and pathological features of 61 formalin-fixed pancreatic cancer specimens and 10 normal pancreases as controls were analyzed. Furthermore, the expression of Protein Gene Product 9.5 (PGP9.5), Myelin P0 protein (MPP), NGF, TrkA, and p75 were examined by immunohistochemistry. The median number of nerves, the median area of neural tissue, and the median nerve diameter per 10 mm2 were larger in the hyperglycemia group than those in the euglycemia group (p = 0.007, p = 0.009, and p = 0.004, respectively). The integrated optical density (IOD) of MPP staining was lower in the hyperglycemia group than those in the euglycemia group (p = 0.019), while the expression levels of NGF and p75 were higher in the hyperglycemia group than those in the euglycemia group (p = 0.002, and p = 0.026, respectively). The nerve bundle invasion of pancreatic cancer was more frequent in the hyperglycemia group than in the euglycemia group (p = 0.000).Conclusions/SignificanceNerve damage and regeneration occur simultaneously in the tumor microenvironment of pancreatic cancer patients with hyperglycemia; the simultaneous occurrence may aggravate the process of perineural invasion. The abnormal expression of NGF and p75 may also be involved in this process and subsequently lead to a lower rate of curative surgery.

Expression of protein gene product 9.5, vasoactive intestinal peptide and neuropeptide Y in the mucosa of the neovagina after sigmoid colon vaginoplasty

To investigate re-innervation in the neovaginal mucosa of patients underwent sigmoid colon vaginoplasty in treatment of Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS). Biopsies in the upper third of the posterior neovagina were taken in 20 patients treated by sigmoid colon vaginoplasty at 1, 2 and 3 years after surgery, respectively. Protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) were detected by immunohistochemical method and compared with those in intact sigmoid colon mucosa. (1) Density of nerve fiber: abundant distribution of PGP 9.5 nerve fibers were observed in the mucosal muscle layer, submucosa, and smooth muscle layer of the neovagina. The nerve fibers of VIP and NPY immunoreactivity were mainly distributed around blood vessels and in the smooth muscles. In the neovagina, the density of nerve fibers of PGP 9.5 of 17 ± 6 were much more than VIP of 2.9 ± 1.0 and NPY of 2.5 ± 0.8 significantly (P < 0.05). (2) Expression of PGP 9.5 in neovagina: at 1 year after surgery, PGP 9.5 positive expression of 14 ± 4 was significantly lower in the neovagina than 28 ± 7 in the intact sigmoid colon (P < 0.05). However, after 2 to 3 years, its expression displayed an upgrade tendency in the neovagina and was significantly higher at the 3 year postoperatively than that at the 1 years postoperatively (22 ± 7 vs. 14 ± 4, P < 0.05). The changes were much more obvious in submucosa. (3) The expression of VIP and NPY in neovagina: at 1 year after surgery, VIP and NPY positive nerve fibers were also decreased in the neovagina when compared with those in the intact sigmoid colon (2.3 ± 0.7 vs. 5.3 ± 1.4, P < 0.05; 2.5 ± 1.1 vs. 5.5 ± 1.1, P < 0.05). At 2 to 3 years after surgery, the positive VIP fiber showed initially decreased and subsequently increased tendency. The density of VIP of 3.7 ± 0.7 in the neovagina at 3 years postoperatively was higher than 2.3 ± 0.7 at 1 years postoperatively (P < 0.05). No significant up-regulation was observed in NPY-positive expression in the neovagina within 3 years after operation. Distribution of sensory PGP 9.5, VIP and NPY immunoreactive nerve fibers was similar to the pattern observed within the intact sigmoid colon wall. The number of nerve fibers in the neovagina decreased after surgery and then increased subsequently within 3 years after surgery.

Cardiovascular autonomic neuropathy in spontaneously diabetic rats with and without application of EGb 761.

Cardiovascular autonomic neuropathy causes abnormalities in the diabetic heart with various clinical sequelae, including exercise intolerance, arrhythmias and painless myocardial infarction. Little is known about (ultra)structural alterations of the myocardial nervous network. On the assumption that this diabetes-specific neuropathy develops due to permanently increased oxidative stress by liberation of oxygen-free radicals, adjuvant application of antioxidative therapeutics appears promising in preventing or delaying long-term diabetic complications. We have investigated the effects of Ginkgo biloba extract (EGb 761), a radical scavenger, against diabetes-induced myocardial nervous damage in spontaneously diabetic BioBreeding/Ottawa Karlsburg (BB/OK) rats. Morphological and morphometric parameters were evaluated by electron microscopy. We used immunohistochemistry to investigate protein expression of protein gene product 9.5, S100 protein, and thyroxin hydroxylase as a neuronal marker. Alterations of cardiac sympathetic activity were measured using the in vivo 123I-metaiodobenzyl-guanidine imaging, and the immunofluorescent labeling of beta1-adrenergic receptors and adenylate cyclase. Our results revealed that A) Diabetes results in slight to moderate ultrastructural alterations (hydrops, disintegration of substructure) of autonomic nerve fibers and related Schwann cells in untreated BB diabetic rats; B) Cardiac sympathetic integrity and activity is impaired due to alterations in the presynaptic nerve terminals and the postsynaptic ß1-AR-AC coupling system; C) Pre-treatment of diabetic myocardium with EGb results in an improvement of most of these parameters compared to unprotected myocardium. In conclusion, EGb may act as a potent therapeutic adjuvant in diabetics with respect to cardiovascular autonomic neuropathy, which may contribute to the prevention of late complications in diabetes.

318 EFFECT OF 6-N-PROPYL-2-THIOURACIL-INDUCED HYPOTHYROIDISM IN THE HOST MOUSE ON THE DEVELOPMENT OF BOVINE TESTIS XENOGRAFTS

In rodents, thyroid hormones inhibit Sertoli cell proliferation, promote Sertoli cell differentiation, and accelerate lumen formation in the seminiferous tubules. Conversely, transient hypothyroidism prolongs Sertoli cell proliferation, leading to increased Sertoli cell number and testicular size. In order to evaluate whether 6-N-propyl-2-thiouracil (PTU)-induced hypothyroidism in the host mouse would affect seminiferous tubule development and germ cell differentiation, and subsequently increase spermatogenesis in bovine testis xenografts, fragments (∼1 mm3) of testes from 1-wk-old Holstein calves (n = 6) were transplanted ectopically to castrated immunodeficient male mice (n = 6/donor). Mice (n = 3/donor) were treated with 0.1% (w/v) PTU in drinking water for 4 weeks or left as control. At 5 and 7 months after grafting, grafts were analyzed by morphometry and immunohistochemistry for expression of protein gene product 9.5 (PGP 9.5) as a germ cell marker, and Mullerian-inhibiting substance (MIS) and androgen receptor (AR) to assess Sertoli cell maturation. For each variable, averages of each group were compared at each collection point by t-test PTU treatment to the drinking water for 1 month suppressed thyroid hormone levels (T4) in host mice without negative systemic effects (0.3 ± 0.2 v. 4 ± 0.3 μg dL-1 at 4 weeks in treated v. control mice, respectively, P &lt; 0.05). Spermatogenesis in recovered grafts was arrested at meiosis regardless of treatment and collection time. Graft weight was lower in treated mice than in controls (21 ± 4 v. 42 ± 5 and 24 ± 9 v. 51 ± 5 mg, at 5 and 7 months, respectively, P &lt; 0.05). Volume density of the tubular and intertubular compartments, and seminiferous epithelium, was not affected by treatment (P &gt; 0.05); however, treatment reduced lumen density compared to controls (9 ± 2 v. 19 ± 3 and 12 ± 1 v. 24 ± 4%) and tubular diameter (121 ± 3 v. 140 ± 7 and 144 ± 2v. 170 ± 2 (im, at 5 and 7 months, respectively (P &lt; 0.05). Tubule length per milligram was not different at 5 months between control and treated groups (P &gt; 0.05) but was increased at 7 months in the treated grafts (50 ± 1 v. 30 ± 1 cm, P &lt; 0.05). Number of Sertoli cells per milligram was not affected by treatment (P &gt; 0.05). However, Sertoli cell volume was increased in controls (440 ± 19 v. 341 ± 14 and 504 ± 6 v. 388 ± 18 μm3, at 5 and 7 months, respectively, P &lt; 0.05). The number of germ cells per 100 Sertoli cells was not different between groups at any collection time (P &gt; 0.05). Sertoli cells showed variable MIS expression and lack of or weak AR expression regardless of treatment and collection time, indicating an immature phenotype. In conclusion, suppression of thyroid hormone levels in host mice affects seminiferous tubule development in bovine testis xenografts, demonstrating that endocrine manipulation of the mouse host will affect xenografts in a predictable manner. However, treatment did not affect number and differentiation of germ cells. Rather, incomplete Sertoli cell maturation appears to lead to incomplete germ cell differentiation in bovine testis xenografts. Supported by USDA (2007-35203-18213).

Effect of capsaicin treatment on nociceptors in rat glabrous skin one day after plantar incision

Dilute capsaicin produces a differential effect on incision-related pain behaviors depending upon the test; it reduces heat hyperalgesia and guarding pain but not mechanical hyperalgesia. This suggests that common mechanisms for heat hyperalgesia and guarding pain occur, and distinct mechanisms exist for mechanical hyperalgesia. The purpose of the present study was to evaluate the effect of capsaicin treatment on the activity of cutaneous nociceptors sensitized by incision to understand the mechanisms for the selective action of dilute capsaicin on incisional pain. We compared the effect of 0.05% capsaicin vs. vehicle treatment on pain behaviors after incision and on the activity of nociceptors from these same rats using the in vitro glabrous skin–nerve preparation. Immunohistochemical expression of protein gene product 9.5 (PGP9.5), neurofilament 200, calcitonin gene related peptide (CGRP) and isolectin B4 (IB4) in skin was also evaluated 1 week after 0.05% capsaicin infiltration. Infiltration of 0.05% capsaicin decreased CGRP and IB4/PGP9.5-immunoreactivity of nociceptors in skin. The same dose of capsaicin that inhibited heat hyperalgesia and guarding behavior interfered with chemo- and heat sensitivity of C-fibers. Neither mechanical hyperalgesia nor mechanosensitivity of nociceptors was affected by capsaicin, suggesting that the concentration of capsaicin used in this study did not cause fiber degeneration. These results demonstrate that nociceptors desensitized by capsaicin contribute to heat hyperalgesia and guarding pain after plantar incision. These putative TRPV1-expressing C-fibers are sensitized to heat and acid after incision, and the transduction of heat and chemical stimuli after plantar incision is impaired by dilute capsaicin.

ANALYSIS OF THE NEURONAL MARKER PROTEIN GENE PRODUCT 9.5 IN INTERNAL LIMITING MEMBRANES AFTER INDOCYANINE-GREEN ASSISTED PEELING

Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.

Accuracy of Combined Protein Gene Product 9.5 and Parafibromin Markers for Immunohistochemical Diagnosis of Parathyroid Carcinoma

Parafibromin, encoded by HRPT2, is the first marker with significant benefit in the diagnosis of parathyroid carcinoma. However, because parafibromin is only involved in up to 70% of parathyroid carcinomas and loss of parafibromin immunoreactivity may not be observed in all cases of HRPT2 mutation, a complementary marker is needed. We sought to determine the efficacy of increased expression of protein gene product 9.5 (PGP9.5), encoded by ubiquitin carboxyl-terminal esterase L1 (UCHL1) as an additional marker to loss of parafibromin immunoreactivity for the diagnosis of parathyroid carcinoma. In total, 146 parathyroid tumors and nine normal tissues were analyzed for the expression of parafibromin and PGP9.5 by immunohistochemistry and for UCHL1 by quantitative RT-PCR. These samples included six hyperparathyroidism-jaw tumor syndrome-related tumors and 24 sporadic carcinomas. In tumors with evidence of malignancy, strong staining for PGP9.5 had a sensitivity of 78% for the detection of parathyroid carcinoma and/or HRPT2 mutation and a specificity of 100%. Complete lack of nuclear parafibromin staining had a sensitivity of 67% and a specificity of 100%. PGP9.5 was positive in a tumor with the HRPT2 mutation L64P that expressed parafibromin. Furthermore, UCHL1 was highly expressed in the carcinoma/hyperparathyroidism-jaw tumor syndrome group compared to normal (P < 0.05) and benign specimens (P < 0.001). These results suggest that positive staining for PGP9.5 has utility as a marker for parathyroid malignancy, with a slightly superior sensitivity (P = 0.03) and similar high specificity to that of parafibromin.

Canine Cutaneous Spindle Cell Tumours with Features of Peripheral Nerve Sheath Tumours: A Histopathological and Immunohistochemical Study

In veterinary medicine, the term peripheral nerve sheath tumour is usually restricted to neoplasms that are closely associated with an identified nerve. Thirty-three cases of canine cutaneous tumours previously classified as spindle cell tumours with features resembling peripheral nerve sheath tumours were examined. Two histological patterns were identified: dense areas of spindle shaped cells resembling the Antoni A pattern and less cellular areas with more pleomorphic cells resembling the Antoni B pattern. Immunohistochemically, all tumours uniformly expressed vimentin and 15/33 (45.4%) had scattered and patchy expression of S-100. Laminin expression was found in 25/33 (75.7%) tumours and collagen IV labelling occurred in 14/33 (42.4%). Expression of protein gene product 9.5 was detected in 31/33 (93.9%) of tumours and neuron specific enolase labelling was present in 27/33 (81.8%). Glial fibrillary acidic protein was only expressed within the cytoplasm of some large multinucleated cells in one tumour. These findings suggest that any cutaneous tumour with one of the two histopathological patterns described above should be described as a cutaneous peripheral nerve sheath tumour and that expression of S-100, laminin and collagen IV may be used to define a schwannoma.