We analysed the motor behaviour of Wistar rats after 7 days lesion in the left striatum, injected with apomorphine (APO) and stimulated by a continuous magnetic field of 3,200 Gauss. For the behaviour assessment, we utilised the activity cage test and the rotarod test. Sixty-eight male Wistar rats were divided into six groups: control, sham, sham magnetic, lesion, and stimulated South and North Poles. After the experiments, coronal sections of the striatum were taken and stained with Nissl for analysis of the lesion. In the activity cage test for distance (F = 3.19), time of activity (F = 5.46) and crossings (F = 3.31) in all groups, except for the North Pole-stimulated group, we observed a significant increase in these behaviours when compared to the control group. Considering the number of counterclockwise turns, we observed a significant increase in the lesion in the South and North Pole stimulation groups compared with the control group. Highlighting the minor number of counterclockwise turns observed in the North Pole-stimulated group in relation to the South Pole-stimulated and Lesion groups (F = 16.01). The rotarod test revealed a decrease in the time spent in this apparatus for the Lesion group when compared to all other groups (F = 5.46). The morphometric analysis showed a reduction in the number of neurons in the Lesion group in relation to all other groups (F = 5.13). Thus, the results suggest that the static magnetic field north and south promoted a distinct behavioural profile and morphological preservation after 7 days of lesion with quinolinic acid associated with APO.

The nervous system is highly plastic during the neonatal period, being sensitive to noxious stimuli, which may cause short- and long-term pain responsivity changes. Understanding plasticity in peripheral pain pathways is crucial, particularly when the nervous system is still under development and remodeling process. Substance P (SP) is widely used as a marker for peripheral neurons with unmyelinated and small myelinated fibers. We investigated the number of SP immunoreactive neurons in the dorsal root ganglion (DRG) of male and female Wistar rats, 15 and 180 days after nociceptive stimulation during the neonatal period. Right and left 5th lumbar (L5) DRG were incubated in rabbit polyclonal anti-substance P primary followed by biotinylated donkey anti-rabbit secondary antibodies. Reaction was revealed with a nickel-diaminobenzidine solution. Labeled neurons were counted and compared between ages, genders and groups. Gender differences were present in both ages, with the number of SP-positive DRG neurons being larger in 15-days-old males on both sides. After 180 days, males showed a larger number of SP-positive neurons than females only on the nociceptive stimulated side. An increased number of SP-positive neurons in the DRG on the stimulated side was present in females, immediately after nociceptive stimulation, but not after 180 days. In conclusion, neonatal noxious stimulation caused a permanent increase in SP-positive DRG neurons in males that was not observed in females, suggesting that differences in pain processing/responsivity between genders could be related to morphological alterations of the nervous system. Anat Rec, 301:849-861, 2018. © 2017 Wiley Periodicals, Inc.

Satellite glial cells (SGCs) are found in the dorsal root ganglia (DRG) surrounding completely and individually sensory neurons, forming a thin sheath cells with regulatory function of the neuronal microenvironment. During the nociceptive stimulus the SGCs are activated and express glial fibrillary acidic protein (GFAP). Sustained nociceptive stimulus may cause morphological changes in SGCs influencing the mechanisms of pain chronification that may differ between genders. Understanding the plasticity in peripheral pain pathways considering gender differences is crucial, particularly when noxious stimuli are applied to neonates, when the nervous system is still under rapid and intense remodeling. We investigated the density (number/area) of neurons in the DRG that were enveloped by GFAP immunoreactive SGCs. Male (N=6 per group) and female (N=6 per group) Wistar rats, 15 and 180 days old after neonatal nociceptive stimulation [1] were used. Right and left L5 DRG were removed, frozen and 8 μm thick criossections were incubated in rabbit polyclonal anti‐GFAP antibody (1:2.000; 24hr). Immunostaining protocol was used as described [2]. The DRG area was measured with the aid of computer software. Neurons enveloped by a ring of GFAP‐labeled SGCs were counted, their density (number/area) was calculated and data were compared between right and left sides, 15 and 180 days of age and genders. Differences were considered significant if p<0.05. There was an increased density of neurons enveloped by a ring of GFAP‐labeled SGCs on both genders, 15 days after the nociceptive stimulus, compared to controls, with females presenting a larger density of these neurons compared to males at this age, on the stimulated side. At age of 180 days, the difference between pain and control groups persisted only in females. The comparison between genders showed that males present a smaller density of neurons enveloped by a ring of GFAP‐labeled SGCs compared to females on both ages with statistical significance only at 15 days old groups. There is an extensive literature clearly pointing to the fact that men and women are different in their responses to pain. Women present increased sensitivity to pain and differ in pain intervention responsivity. Despite that sociocultural and psychological factors certainly play a role in these gender differences, evidence from animals studies reveal important, qualitative and quantitative differences at low levels of the neuroaxis. Our results add extra evidence that multiple biological factors and mechanisms underlie gender differences in sensitivity and responses to pain.Support or Funding InformationFinancial support: FAPESP, CNPq, CAPES and FAEPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Peripheral nerve biopsy is an invaluable aid to diagnosis carefully selected patients. The nerves histological evaluation gives clues for investigating diseases mechanisms and causes, as well as guides the therapeutic planning of inflammatory, infectious, demyelinating or degenerative lesions [1]. Nerves histological processing method that provides the best image quality is the epoxy resin embedding. Thus, nerve sections can be observed by light and/or transmission electron microscopy (TEM) and classes of nerve fibers can be morphometrically identified and studied. Epoxy resin embedding and TEM is considered the gold standard method for unmyelinated fibers identification and quantification. More recently, the immunohistochemistry technique (IHC) is being added to the inflammatory neuropathy investigation and the most important of the panel of antibodies employed are those needed to differentiate lymphocytes into T (CD3þve) and B cells (CD20þve). In addition, antibodies against macrophages, myelin basic protein, neurofilaments, and epithelial membrane antigen (EMA) (for perineurial cells) are included [1]. Neurotransmitters and their fiber “carrier” are much less explored. We suggest that it would be possible to use the IHC to identify nerve fiber classes as an alternative to TEM and morphometry, with the gain on functional information. To investigate this hypothesis we used IHQ and immunofluorescence (IF) to immunolabel small myelinated and unmyelinated fibers in sural nerves of rats. The nerves were surgically removed, immersed in paraformaldehyde 4% for 18 hours, and cryoprotected in increasing concentrations of sucrose solutions, before transversal cryosectioning (12μm). For IHC, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:5.000, 18h) followed by anti‐rabbit IgG (1:500; 2h) and Extravidin‐HRP (1:1500, 2h). A reaction with diaminobenzidine intensified with nickel revealed immunoreactivity, observed by light microscopy. For IF, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:1.000, 18h) followed by anti‐rabbit IgG (Alexa Fluor® 594) (1:200, 2h). The immunoreactivity was observed by fluorescence microscopy. Both protocols worked on transverse sections to identify substance P immunolabeled small myelinated and unmyelinated fibers. Results were compared with transverse sections prepared for TEM and the IHC results showed labeled small myelinated and unmyelinated axons intermingled with large unlabeled myelinated axons, with preservation of their myelin sheath. The IHC transverse sections were comparable to the epoxy resin embedded for small fiber localization in the endoneural space. Further studies on quantification of these fibers will be performed comparing both methods (IHC and IF) with the TEM images in order to show that IHC might also be a reliable method for quantifying axons.Support or Funding InformationFinancial support: FAPESP, CNPq, CAPES and FAEPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A hipertensão arterial sistêmica é uma doença altamente incidente e fator de risco, principalmente, para acidentes vasculares cerebrais. No entanto, é raramente explorada como possível causa de neuropatia para fibras mielínicas finas e amielínicas. A biópsia do nervo periférico é um recurso importante no diagnóstico de neuropatias, sendo inclusão em resina epóxi padrão-ouro de identificação e quantificação de fibras nervosas. No entanto, a imunomarcação vem sendo adicionada à investigação de neuropatias inflamatórias. Porém, anticorpos contra neurotransmissores para identificação de fibras específicas são muito pouco explorados. Assim, o objetivo deste estudo foi identificar substância P (SP), através de imunohistoquímica (IHQ), e quantificar a densidade de fibras mielínicas finas e amielínicas reativas a este neurotransmissor nos segmentos proximais e distais dos nervos surais de ratos espontaneamente hipertensos (SHR) e normotensos (WKY), machos e fêmeas (n=6 por grupo) com 20 semanas de idade, comparando os diferentes segmentos e lados deste nervo. Após anestesia e medida direta da pressão arterial, os nervos foram dissecados e preparados tecnicamente para IHQ, a qual seguiu protocolo padronizado. A quantificação da densidade de fibras reativas à SP presente nos nervos foi realizada através do programa computacional Image J. Para dados pareados com distribuição normal realizou-se teste t de Student pareado, em caso contrário foi executado o teste de Wilcoxon, e para dados não pareados com distribuição normal realizou-se teste t de Student, em caso contrário, o teste de Mann-Whitney. Os resultados foram considerados estatisticamente significativos quando p<0,05. Diferenças significativas foram observadas para o parâmetro proposto quando comparadas as fêmeas WKY com as SHR e quando comparados os machos WKY com os SHR. Os resultados indicaram que a hipertensão arterial sistêmica altera importantes componentes do nervo Sural de animais hipertensos. Além disso, as comparações entre machos e fêmeas WKY e SHR permitiram concluir também que a densidade das fibras imunomarcadas neste nervo não possui correlação com gênero, mas sim com linhagem. Palavras-chave: Nervo Sural. Fibras mielínicas finas. Fibras amielínicas. Substância P. Ratos espontaneamente hipertensos. Gêneros. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Código de Financiamento 88882.328289/2019-01.