A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death, gliosis, and electroretinogram distortion in male rats subjected to perinatal asphyxia.

Introduction: Perinatal asphyxia (PA) represents a major problem in perinatology and may cause visual losses, including blindness. We, and others, have shown that hypothermia prevents retinal symptoms associated to PA. In the present work, we evaluate whether a hypothermia mimetic small molecule, zr17-2, has similar effects in the context of PA. Methods: Four experimental groups were studied in male rats: Naturally born rats as controls (CTL), naturally born rats injected s.c. with 50 µL of 330 nmols/L zr17-2 (ZR), animals that were exposed to PA for 20 min at 37°C (PA), and rats that were exposed to PA and injected with zr17-2 (PA-ZR). Forty-five days after treatment, animals were subjected to electroretinography. In addition, morphological techniques (TUNEL, H&E, multiple immunofluorescence) were applied to the retinas. Results: A reduction in the amplitude of the a- and b-wave and oscillatory potentials (OP) of the electroretinogram (ERG) was detected in PA animals. Treatment with zr17-2 resulted in a significant amelioration of these parameters (p < 0.01). In PA animals, a large number of apoptotic cells was found in the GCL. This number was significantly reduced by treatment with the small molecule (p < 0.0001). In a similar way, the thickness of the inner retina and the intensity of GFAP immunoreactivity (gliosis) increased in PA retinas (p < 0.0001). These parameters were corrected by the administration of zr17-2 (p < 0.0001). Furthermore, injection of the small molecule in the absence of PA did not modify the ERG nor the morphological parameters studied, suggesting a lack of toxicity. Discussion: In conclusion, our results indicate that a single s.c. injection of zr17-2 in asphyctic neonates may provide a novel and efficacious method to prevent the visual sequelae of PA.

A hypothermia mimetic molecule (zr17-2) reduces ganglion cell death and electroretinogram distortion in a rat model of intraorbital optic nerve crush (IONC).

Introduction: Ocular and periocular traumatisms may result in loss of vision. Our previous work showed that therapeutic hypothermia prevents retinal damage caused by traumatic neuropathy. We also generated and characterized small molecules that elicit the beneficial effects of hypothermia at normal body temperature. Here we investigate whether one of these mimetic molecules, zr17-2, is able to preserve the function of eyes exposed to trauma. Methods: Intraorbital optic nerve crush (IONC) or sham manipulation was applied to Sprague-Dawley rats. One hour after surgery, 5.0 µl of 330 nmol/L zr17-2 or PBS, as vehicle, were injected in the vitreum of treated animals. Electroretinograms were performed 21 days after surgery and a- and b-wave amplitude, as well as oscillatory potentials (OP), were calculated. Some animals were sacrificed 6 days after surgery for TUNEL analysis. All animal experiments were approved by the local ethics board. Results: Our previous studies showed that zr17-2 does not cross the blood-ocular barrier, thus preventing systemic treatment. Here we show that intravitreal injection of zr17-2 results in a very significant prevention of retinal damage, providing preclinical support for its pharmacological use in ocular conditions. As previously reported, IONC resulted in a drastic reduction in the amplitude of the b-wave (p < 0.0001) and OPs (p < 0.05), a large decrease in the number of RGCs (p < 0.0001), and a large increase in the number of apoptotic cells in the GCL and the INL (p < 0.0001). Interestingly, injection of zr17-2 largely prevented all these parameters, in a very similar pattern to that elicited by therapeutic hypothermia. The small molecule was also able to reduce oxidative stress-induced retinal cell death in vitro. Discussion: In summary, we have shown that intravitreal injection of the hypothermia mimetic, zr17-2, significantly reduces the morphological and electrophysiological consequences of ocular traumatism and may represent a new treatment option for this cause of visual loss.

First record of an infection by tissue cyst-forming coccidia in wild vizcachas (Lagostomus maximus, Rodentia) of Argentina.

Endoparasites of the Sarcocystidae family share the ability to form tissue cysts in their intermediate hosts, ultimately leading to pathogenesis in the definitive hosts that include various mammals, reptiles and birds. In our research on the endocrinology of the female vizcachas (Lagostomus maximus), we have found abnormal cystic structures in the ovaries of some individuals. So far, no cases of infection by tissue cyst-forming parasites have been reported in this species. To evaluate whether this autochthonous wild rodent is an intermediate host of an undescribed endoparasite, histological sections from various organs were examined. Pinhead-sized tissue cysts were found in the ovaries, mammary glands, uterus, pituitary, brain, adrenals and spleen, of both pregnant and non-pregnant females. The presence of cysts in the adult brain and embryonic tissue is indicative of the ability of the parasite to cross both the blood-brain and placental barriers. The infected brains exhibited a lower cyst density than that seen in other organs. Regardless of their location in superficial or deep tissue, the cysts were surrounded by a layer of connective tissue. Histologically, the cyst wall consisted of an outer layer of fibroblasts and collagen fibers, and an inner, granular-looking layer composed of host nucleated cells surrounding thousands of spindle-shaped bradyzoites. Outside the cysts, the host cellular structures showed normal appearance. The remarkable morphological similarities between the cysts studied here with those reported in naturally infected rabbits from an area neighboring the one inhabited by the vizcachas point to Besnoitia sp. as a plausible candidate. More studies will be necessary to confirm the identity of the parasite. Nevertheless, this is the first report of L. maximus as an intermediate host for a tissue cyst-forming coccidia.

Hypothermic Shock Applied After Perinatal Asphyxia Prevents Retinal Damage in Rats.

Perinatal asphyxia (PA) can cause retinopathy and different degrees of visual loss, including total blindness. In a rat model of PA, we have previously shown a protective effect of hypothermia on the retina when applied simultaneously with the hypoxic insult. In the present work, we evaluated the possible protective effect of hypothermia on the retina of PA rats when applied immediately after delivery. Four experimental groups were studied: Rats born naturally as controls (CTL), animals that were exposed to PA for 20 min at 37°C (PA), animals exposed to PA for 20 min at 15°C (HYP), and animals that were exposed to PA for 20 min at 37°C and, immediately after birth, kept for 15 min at 8°C (HYP-PA). To evaluate the integrity of the visual pathway, animals were subjected to electroretinography at 45 days of age. Molecular (real time PCR) and histological (immunohistochemistry, immunofluorescence, TUNEL assay) techniques were applied to the eyes of all experimental groups collected at 6, 12, 24, and 48 h, and 6 days after birth. PA resulted in a significant reduction in the amplitude of the a- and b-wave and oscillatory potentials (OP) of the electroretinogram. All animals treated with hypothermia had a significant correction of the a-wave and OP, but the b-wave was fully corrected in the HYP group but only partially in the HYP-PA group. The number of TUNEL-positive cells increased sharply in the ganglion cell layer of the PA animals and this increase was significantly prevented by both hypothermia treatments. Expression of the cold-shock proteins, cold-inducible RNA binding protein (CIRP) and RNA binding motif protein 3 (RBM3), was undetectable in retinas of the CTL and PA groups, but they were highly expressed in ganglion neurons and cells of the inner nuclear layer of the HYP and HYP-PA groups. In conclusion, our results suggest that a post-partum hypothermic shock could represent a useful and affordable method to prevent asphyxia-related vision disabling sequelae.

Methylene Blue Prevents Retinal Damage Caused by Perinatal Asphyxia in the Rat.

Perinatal asphyxia (PA) is responsible for a large proportion of neonatal deaths and numerous neurological sequelae, including visual dysfunction and blindness. In PA, the retina is exposed to ischemia/reoxygenation, which results in nitric oxide (NO) overproduction and neurotoxicity. We hypothesized that methylene blue (MB), a guanylyl cyclase inhibitor, and free-radical scavenger currently used in the clinic, may block this pathway and prevent PA-induced retinal degeneration. Male rat pups were subjected to an experimental model of PA. Four groups were studied: normally delivered (CTL), normally delivered treated with 2 mg Kg-1 MB (MB), exposed to PA for 20 min at 37°C (PA), and exposed to PA and, then, treated with MB (PA-MB). Scotopic electroretinography performed 45 days after birth showed that PA animals had significant defects in the a- and b-waves and oscillatory potentials (OP). The same animals presented a significant increase in the thickness of the inner retina and a large number of TUNEL-positive cells. All these physiological and morphological parameters were significantly prevented by the treatment with MB. Gene expression analysis demonstrated significant increases in iNOS, MMP9, and VEGF in the eyes of PA animals, which were prevented by MB treatment. In conclusion, MB regulates key players of inflammation, matrix remodeling, gliosis, and angiogenesis in the eye and could be used as a treatment to prevent the deleterious visual consequences of PA. Given its safety profile and low cost, MB may be used clinically in places where alternative treatments may be unavailable.

The key action of estradiol and progesterone enables GnRH delivery during gestation in the South American plains vizcacha, Lagostomus maximus.

The South American plains vizcacha, Lagostomus maximus, is the only mammal described so far that shows expression of estrogen receptors (ERs) and progesterone receptors (PRs) in gonadotropin-releasing hormone (GnRH) neurons. This animal therefore constitutes an exceptional model for the study of the effect of steroid hormones on the modulation of the hypothalamic-pituitary-ovarian (HPO) axis. By using both in vivo and ex vivo approaches, we have found that pharmacological doses of progesterone (P4) and estradiol (E2) produced an inhibition in the expression of hypothalamic GnRH, while physiological doses produced a differential effect on the pulsatile release frequency or genomic expression of GnRH. Our ex vivo experiment indicates that a short-term effect of E2 modulates the frequency of GnRH release pattern that would be associated with membrane ERs. On the other hand, our in vivo approach suggests that a long-term effect of E2, acting through the classical nuclear ERs-PRs pathway, would produce the modification of GnRH mRNA expression during the GnRH pre-ovulatory surge. Particularly, P4 induced a rise in GnRH mRNA expression and protein release with a decrease in its release frequency. These results suggest different levels of action of steroid hormones on GnRH modulation. We conclude that the fine action of E2 and P4 constitute the key factor to enable the hypothalamic activity during the pregnancy of this mammal.

Multiple failures in the lutenising hormone surge generating system in GABAB1KO female mice.

Female mice lacking GABAB receptors, GABAB1KO, show disrupted oestrous cycles, reduced pregnancies and increased hypothalamic Gnrh1 mRNA expression, whereas anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) Kiss1 mRNA was not affected. In the present study, we characterise the important components of the gonadotrophic preovulatory surge, aiming to unravel the origin of this reproductive impairment. In GABAB1KO and wild-type (WT) females, we determined: (i) hypothalamic oestrogen receptor (ER)α and ß and aromatase mRNA and protein expression; (ii) ovulation index and oestrus serum follicle-stimulating hormone (FSH) and pituitary Gnrh1r expression; (iii) in ovariectomised-oestradiol valerate-treated mice, we evaluated ex vivo hypothalamic gonadotrophin-releasing hormone (GnRH) pulsatility in the presence/absence of kisspeptin (Kiss-10, constant or pulsatile) and oestradiol (constant); and (iv) in ovariectomised-oestradiol silastic capsule-treated mice (proestrous-like environment), we evaluated morning and evening kisspeptin neurone activation (c-Fos+) and serum luteinising homrone (LH). In the medial basal hypothalamus of oestrus GABAB1KOs, aromatase and ERα mRNA and protein were increased, whereas ERß was decreased. In GABAB1KOs, the ovulation index was decreased together with decreased first oestrus serum FSH and increased pituitary Gnrh1r mRNA. Under constant Kiss-10 stimulation, hypothalamic GnRH pulse frequency did not vary, although GnRH mass/pulse was increased in GABAB1KOs. In WTs, pulsatile Kiss-10 together with constant oestradiol significantly increased GnRH pulsatility, whereas, in GABAB1KOs, oestradiol alone increased GnRH pulsatility and this was reversed by pulsatile Kiss-10 addition. In GABAB1KOs AVPV/PeN kisspeptin neurones were similarly activated (c-Fos+) in the morning and evening, whereas WTs showed the expected, marked evening stimulation. LH correlated with activated kisspeptin cells in WT mice, whereas GABAB1KO mice showed high, similar LH levels both in the morning and evening. Taken together, all of these alterations point to impairment in the trigger of the preovulatory GnRH surge that entails the reproductive alterations described.

Local production of neurostradiol affects gonadotropin-releasing hormone (GnRH) secretion at mid-gestation in Lagostomus maximus (Rodentia, Caviomorpha).

Females of the South American plains vizcacha, Lagostomus maximus, show peculiar reproductive features such as massive polyovulation up to 800 oocytes per estrous cycle and an ovulatory process around mid-gestation arising from the reactivation of the hypothalamic-hypophyseal-ovary (H.H.O.) axis. Estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) expression. Biosynthesis of estrogens results from the aromatization of androgens by aromatase, which mainly occurs in the gonads, but has also been described in the hypothalamus. The recently described correlation between GnRH and ERα expression patterns in the hypothalamus of the vizcacha during pregnancy, with coexpression in the same neurons of the medial preoptic area, suggests that hypothalamic synthesis of E2 may affect GnRH neurons and contribute with systemic E2 to modulate GnRH delivery during the gestation. To elucidate this hypothesis, hypothalamic expression and the action of aromatase on GnRH release were evaluated in female vizcachas throughout pregnancy. Aromatase and GnRH expression was increased significantly in mid-pregnant and term-pregnant vizcachas compared to early-pregnant and nonpregnant females. In addition, aromatase and GnRH were colocalized in neurons of the medial preoptic area of the hypothalamus throughout gestation. The blockage of the negative feedback of E2 induced by the inhibition of aromatase resulted in a significant increment of GnRH-secreted mass by hypothalamic explants. E2 produced in the same neurons as GnRH may drive intracellular E2 to higher levels than those obtained from systemic circulation alone. This may trigger for a prompt GnRH availability enabling H.H.O. activity at mid-gestation with ovulation and formation of accessory corpora lutea with steroidogenic activity that produce the necessary progesterone to maintain gestation to term and guarantee the reproductive success.

Hypothermia Prevents Retinal Damage Generated by Optic Nerve Trauma in the Rat.

Ocular and periocular traumatisms may result in loss of vision. Hypothermia provides a beneficial intervention for brain and heart conditions and, here, we study whether hypothermia can prevent retinal damage caused by traumatic neuropathy. Intraorbital optic nerve crush (IONC) or sham manipulation was applied to male rats. Some animals were subjected to hypothermia (8 °C) for 3 h following surgery. Thirty days later, animals were subjected to electroretinography and behavioral tests. IONC treatment resulted in amplitude reduction of the b-wave and oscillatory potentials of the electroretinogram, whereas the hypothermic treatment significantly (p < 0.05) reversed this process. Using a descending method of limits in a two-choice visual task apparatus, we demonstrated that hypothermia significantly (p < 0.001) preserved visual acuity. Furthermore, IONC-treated rats had a lower (p < 0.0001) number of retinal ganglion cells and a higher (p < 0.0001) number of TUNEL-positive cells than sham-operated controls. These numbers were significantly (p < 0.0001) corrected by hypothermic treatment. There was a significant (p < 0.001) increase of RNA-binding motif protein 3 (RBM3) and of BCL2 (p < 0.01) mRNA expression in the eyes exposed to hypothermia. In conclusion, hypothermia constitutes an efficacious treatment for traumatic vision-impairing conditions, and the cold-shock protein pathway may be involved in mediating the beneficial effects shown in the retina.

Cold Shock Proteins Are Expressed in the Retina Following Exposure to Low Temperatures.

Hypothermia has been proposed as a therapeutic intervention for some retinal conditions, including ischemic insults. Cold exposure elevates expression of cold-shock proteins (CSP), including RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), but their presence in mammalian retina is so far unknown. Here we show the effects of hypothermia on the expression of these CSPs in retina-derived cell lines and in the retina of newborn and adult rats. Two cell lines of retinal origin, R28 and mRPE, were exposed to 32°C for different time periods and CSP expression was measured by qRT-PCR and Western blotting. Neonatal and adult Sprague-Dawley rats were exposed to a cold environment (8°C) and expression of CSPs in their retinas was studied by Western blotting, multiple inmunofluorescence, and confocal microscopy. RBM3 expression was upregulated by cold in both R28 and mRPE cells in a time-dependent fashion. On the other hand, CIRP was upregulated in R28 cells but not in mRPE. In vivo, expression of CSPs was negligible in the retina of newborn and adult rats kept at room temperature (24°C). Exposure to a cold environment elicited a strong expression of both proteins, especially in retinal pigment epithelium cells, photoreceptors, bipolar, amacrine and horizontal cells, Müller cells, and ganglion cells. In conclusion, CSP expression rapidly rises in the mammalian retina following exposure to hypothermia in a cell type-specific pattern. This observation may be at the basis of the molecular mechanism by which hypothermia exerts its therapeutic effects in the retina.

Methylene blue prevents retinal damage in an experimental model of ischemic proliferative retinopathy.

Perinatal asphyxia induces retinal lesions, generating ischemic proliferative retinopathy, which may result in blindness. Previously, we showed that the nitrergic system was involved in the physiopathology of perinatal asphyxia. Here we analyze the application of methylene blue, a well-known soluble guanylate cyclase inhibitor, as a therapeutic strategy to prevent retinopathy. Male rats (n = 28 per group) were treated in different ways: 1) control group comprised born-to-term animals; 2) methylene blue group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery; 3) perinatal asphyxia (PA) group comprised rats exposed to perinatal asphyxia (20 min at 37°C); and 4) methylene blue-PA group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery, and then the pups were subjected to PA as above. For molecular studies, mRNA was obtained at different times after asphyxia, and tissue was collected at 30 days for morphological and biochemical analysis. Perinatal asphyxia produced significant gliosis, angiogenesis, and thickening of the inner retina. Methylene blue treatment reduced these parameters. Perinatal asphyxia resulted in a significant elevation of the nitrergic system as shown by NO synthase (NOS) activity assays, Western blotting, and (immuno)histochemistry for the neuronal isoform of NOS and NADPH-diaphorase activity. All these parameters were also normalized by the treatment. In addition, methylene blue induced the upregulation of the anti-angiogenic peptide, pigment epithelium-derived factor. Application of methylene blue reduced morphological and biochemical parameters of retinopathy. This finding suggests the use of methylene blue as a new treatment to prevent or decrease retinal damage in the context of ischemic proliferative retinopathy.

IGF1 regulation of BOULE and CDC25A transcripts via a testosterone-independent pathway in spermatogenesis of adult mice.

The Deleted in AZoospermia (DAZ) gene family plays an essential role in spermatogenesis and fertility in mammals. This gene family contains two autosomal genes, BOULE and DAZL (DAZ-Like), and the DAZ gene cluster in the Y chromosome. CDC25A (a cell cycle regulator) has been proposed as a putative substrate for the RNA-binding proteins of DAZ family. However, mechanisms regulating DAZ gene expression have been poorly investigated. We analyzed immunohistochemical localization of DAZL, BOULE and CDC25A, as well as the involvement of testosterone (T) and insulin-like growth factor 1 (IGF1) in the modulation of mRNA expression for DAZL, BOULE and CDC25A in the adult mouse testes. It was found that DAZL was mostly immunolocalized in spermatogonia, while BOULE and CDC25A were detected in spermatocytes and round spermatids. Three-color immunofluorescence showed that DAZL-positive cells also expressed proliferating cell nuclear antigen (PCNA). In vitro incubation of the testes showed that neither T nor IGF1 affected DAZL mRNA expression. However, either T or IGF1 increased BOULE mRNA expression. Antiandrogen flutamide abolished the T-induced increase in BOULE mRNA, but had no effect on the IGF1 induced increase in the mouse testes. Extracellular-signal-regulated kinase 1/2 (ERK1/2) inhibitor, U0126, prevented IGF1-induction of BOULE mRNA. It was found that IGF1 increased CDC25A mRNA expression and that U0126 - but not flutamide - abolished the IGF1-induced CDC25A mRNA expression. These results showed that IGF1 regulated the expression of BOULE and CDC25A mRNAs via ERK1/2 signaling and in T-independent pathway during spermatogenesis in the adult mouse testes.

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus).

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERß. Yet throughout pregnancy, ERα and ERß levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.

Plaque-associated overexpression of insulin-degrading enzyme in the cerebral cortex of aged transgenic tg2576 mice with Alzheimer pathology.

It was proposed that insulin-degrading enzyme (IDE) participates in the clearance of amyloid beta (Abeta) in the brain, and its low expression or activity may be relevant for the progression of Alzheimer disease. We performed a longitudinal study of brain level, activity, and distribution of IDE in transgenic mice (Tg2576) expressing the Swedish mutation in human Abeta precursor protein. At 16 months of age, Tg2576 showed a significant 2-fold increment in IDE protein level as compared with 4.5- and 11-month-old animals. The peak of IDE was in synchrony with the sharp accumulation of sodium dodecyl sulfate-soluble Abeta and massive Abeta deposition into plaques. At this stage, IDE appeared surrounding Abeta fibrillar deposits within glial fibrillar acidic protein-positive astrocytes, suggesting that it was locally overexpressed during the Abeta-mediated inflammation process. When primary astrocytes were exposed to fibrillar Abeta in vitro, IDE protein level increased as compared with control, and this effect was reduced by the addition of U0126, a specific inhibitor of the ERK1/2 mitogen-activated protein kinase cascade. We propose that in Tg2576 mice and in contrast to its behavior in Alzheimer brains, active IDE increases with age around plaques as a component of astrocyte activation as a result of Abeta-triggered inflammation.

Age-related changes of the GABA-B receptor in the lumbar spinal cord of male rats and penile erection.

Dorsal horn neurons of lumbosacral spinal cord innervate penile vasculature and regulate penile erection. GABAergic system is involved in the regulation of male sexual behavior. Because aging is frequently accompanied by a progressive decline in erectile function, the aim of this work was to examine age-related changes of the GABA-B receptor in the lumbar spinal cord. Sprague-Dawley rats of 10 and 21 days old, 3, 9 and 20 months old were used. GABA-B receptors were evaluated by quantitative autoradiography using [3H]-Baclofen as ligand with or without GABA (10 microM) to determine the non-specific binding. Ten days after birth a homogeneous neuroanatomical distribution pattern was found in the gray matter, however at 20-day-old adult distribution emerged becoming heterogeneous with the highest binding values at layers II-III and X. In dorsal layers a significant decrease was observed in 9-month-old rats while layer X showed an earlier decrease (21-day-old). GABA-B receptor affinity showed significant age-dependent and regional increase. The GABA-B receptor decrease in aged rats seems not to be related to this receptor inhibitory function in penile erection. Moreover the changes found in GABA-B receptor binding anatomical distribution may indicate its role in the morphological development of the lumbar spinal cord rather than in the decline of the erectile function.

Reduction of the spinal nucleus of the bulbocavernosous volume by experimental diabetes.

The sexually dimorphic nuclei, spinal nucleus of the bulbocavernosus (SNB) and dorsolateral nucleus, are located at the lumbar segment of the rat spinal cord. These nuclei innervate perineal muscles involved in penile erection and ejaculation. Testosterone levels modulate their size in adult male rats. Because diabetes is associated with low levels of testosterone, we have evaluated morphological changes on spinal cord of diabetic animals. Significant reduction in the SNB volume was observed 4 weeks after diabetes induction accomplished by a reduction on the motoneuronal size. Insulin prevents the morphological alterations. No significant changes were observed on other dimorphic nucleus. The altered sexual behavior of diabetic rats could be consequence of the detected reduction in the SNB volume.