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.

Previous results from our laboratory [1] showed that 50% of the aortic depressor nerves (ADN), the afferent arm of the baroreflex, show alterations in early stages of induced diabetes in rats. The recurrent laryngeal nerves (RLN) are known to contain barorreceptor fibers as well and there are no reports on the morphology of the RLN in short term diabetes. Male Wistar rats received a single intravenous streptozotocin injection and control rats received vehicle. On the 15th day after the injections, rats were anesthetized for arterial pressure (AP) and heart rate (HR) recordings, prior to RLN removal. Proximal and distal segments of the right and left RLN were prepared for light microscopy morphometry, carried out with the aid of a computer software. Body weight increased significantly on the control s(309 ± 9 g) compared to the diabetics (200 ± 4 g). As expected, blood glucose levels were significantly high in the diabetic animals. Both the AP and HR were reduced in the diabetic group, attaining significant difference on the HR values. Fascicle morphometry parameters were not different between groups. We suggest that 15 days was not long enough to trigger detectable differences in the RLN fascicles. This difference with our previous findings in the ADN is probably due to the nerve size, since the ADN is much smaller than the RLN.

Despite some reports on the peripheral nerve morphology in spontaneously hypertensive rats (SHR), no investigations on the recurrent laryngeal nerve (RLN), known to contain baroreceptor fibers is available in this rat strain. We aimed to describe the normal morphological and morphometric parameters of the recurrent laryngeal nerves in developing SHR. Animals were divided into three groups: Female SHR 5, 8 and 20 week‐old (n=4 in each group). The animals were anesthetized for direct measurement of blood pressure, perfused with a fixative solution and had their right and left RLN, dissected and prepared for light microscopy and morphometry. Body weight increased significantly with aging. Mean arterial pressure was significantly higher in 20‐week old animals compared to 5 week‐old, but not different between 5 and 8‐week old animals or 8 and 20‐week old ones. In general, proximal segments are larger than distal segments with a significant reduction on the myelinated fiber number from proximal to distal. Myelinated fiber size is larger on the older animals, following the increase in body weight. No significant alterations of the nerve morphology were observed in 20‐week old animals, despite the elevated arterial pressure.Grant Funding Source: FAPESP, CNPq, CAPES and FAEPA