Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18% (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87%, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91% over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

Effects of strain and age on ear wound healing and regeneration in mice

Round holes in the ears of MRL mice tend to close with characteristics of regeneration believed to be absent in other mouse strains (e.g., C57BL/6). We evaluated the kinetics and the histopathology of ear wound closure in young (8 weeks old) C57BL/6 and BALB/c mice. We also used middle-aged (40 weeks old) C57BL/6 mice to evaluate the influence of aging on this process. A circular through-and-through hole was made in the ear, photographs were taken at different times after injury and wound area was measured with digital analysis software. The percentages of closed area measured on day 100 were: 23.57 ± 8.66% for young BALB/c mice, 56.47 ± 7.39% for young C57BL/6 mice, and 75.31 ± 23.65% for middle-aged C57BL/6 mice. Mice were sacrificed on days 1, 3, 5, 25, 44, and 100 for histological evaluation with hematoxylin and eosin, Gomori’s trichrome, periodic acid-Schiff, or picrosirius red staining. In young mice of both strains, healing included re-epithelialization, chondrogenesis, myogenesis, and collagen deposition. Young C57BL/6 and BALB/c mice differed in the organization of collagen fibers visualized using picrosirius-polarization. Sebaceous glands and hair follicles regenerated and chondrogenesis was greater in young C57BL/6 mice. In middle-aged C57BL/6 mice all aspects of regeneration were depressed. The characteristics of regeneration were present during ear wound healing in both young BALB/c and young C57BL/6 mice although they differed in intensity and pattern. Greater ear wound closure in middle-aged C57BL/6 mice was not correlated with regeneration.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Transplantation of muscle-derived stem cells plus biodegradable fibrin glue restores the urethral sphincter in a pudendal nerve-transected rat model

We investigated whether fibrin glue (FG) could promote urethral sphincter restoration in muscle-derived stem cell (MDSC)-based injection therapies in a pudendal nerve-transected (PNT) rat, which was used as a stress urinary incontinence (SUI) model. MDSCs were purified from the gastrocnemius muscles of 4-week-old inbred female SPF Wistar rats and labeled with green fluorescent protein. Animals were divided into five groups (N = 15): sham (S), PNT (D), PNT+FG injection (F), PNT+MDSC injection (M), and PNT+MDSC+FG injection (FM). Each group was subdivided into 1- and 4-week groups. One and 4 weeks after injection into the proximal urethra, leak point pressure (LPP) was measured to assess urethral resistance function. Histology and immunohistochemistry were performed 4 weeks after injection. LPP was increased significantly in FM and M animals after implantation compared to group D (P < 0.01), but was not different from group S. LPP was slightly higher in the FM group than in the M group but there was no significant difference between them at different times. Histological and immunohistochemical examination demonstrated increased numbers of surviving MDSCs (109 ± 19 vs 82 ± 11/hpf, P = 0.026), increased muscle/collagen ratio (0.40 ± 0.02 vs 0.34 ± 0.02, P = 0.044), as well as increased microvessel density (16.9 ± 0.6 vs 14.1 ± 0.4/hpf, P = 0.001) at the injection sites in FM compared to M animals. Fibrin glue may potentially improve the action of transplanted MDSCs to restore the histology and function of the urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin glue may provide a novel cellular therapy method for SUI.

Inactivation of capsaicin-sensitive nerves reduces pulmonary remodeling in guinea pigs with chronic allergic pulmonary inflammation

Pulmonary remodeling is an important feature of asthma physiopathology that can contribute to irreversible changes in lung function. Although neurokinins influence lung inflammation, their exact role in the extracellular matrix (ECM) remodeling remains to be determined. Our objective was to investigate whether inactivation of capsaicin-sensitive nerves modulates pulmonary ECM remodeling in animals with chronic lung inflammation. After 14 days of capsaicin (50 mg/kg, sc) or vehicle administration, male Hartley guinea pigs weighing 250-300 g were submitted to seven inhalations of increasing doses of ovalbumin (1, 2.5, and 5 mg/mL) or saline for 4 weeks. Seventy-two hours after the seventh inhalation, animals were anesthetized and mechanically ventilated and the lung mechanics and collagen and elastic fiber content in the airways, vessels and lung parenchyma were evaluated. Ovalbumin-exposed animals presented increasing collagen and elastic fiber content, respectively, in the airways (9.2 ± 0.9; 13.8 ± 1.2), vessels (19.8 ± 0.8; 13.4 ± 0.5) and lung parenchyma (9.2 ± 0.9; 13.8 ± 1.2) compared to control (P < 0.05). Capsaicin treatment reduced collagen and elastic fibers, respectively, in airways (1.7 ± 1.1; 7.9 ± 1.5), vessels (2.8 ± 1.1; 4.4 ± 1.1) and lung tissue (2.8 ± 1.1; 4.4 ± 1.1) of ovalbumin-exposed animals (P < 0.05). These findings were positively correlated with lung mechanical responses to antigenic challenge (P < 0.05). In conclusion, inactivation of capsaicin-sensitive nerve fibers reduces pulmonary remodeling, particularly collagen and elastic fibers, which contributes to the attenuation of pulmonary functional parameters.

Percutaneous sciatic nerve block with tramadol induces analgesia and motor blockade in two animal pain models

Local anesthetic efficacy of tramadol has been reported following intradermal application. Our aim was to investigate the effect of perineural tramadol as the sole analgesic in two pain models. Male Wistar rats (280-380 g; N = 5/group) were used in these experiments. A neurostimulation-guided sciatic nerve block was performed and 2% lidocaine or tramadol (1.25 and 5 mg) was perineurally injected in two different animal pain models. In the flinching behavior test, the number of flinches was evaluated and in the plantar incision model, mechanical and heat thresholds were measured. Motor effects of lidocaine and tramadol were quantified and a motor block score elaborated. Tramadol, 1.25 mg, completely blocked the first and reduced the second phase of the flinching behavior test. In the plantar incision model, tramadol (1.25 mg) increased both paw withdrawal latency in response to radiant heat (8.3 ± 1.1, 12.7 ± 1.8, 8.4 ± 0.8, and 11.1 ± 3.3 s) and mechanical threshold in response to von Frey filaments (459 ± 82.8, 447.5 ± 91.7, 320.1 ± 120, 126.43 ± 92.8 mN) at 5, 15, 30, and 60 min, respectively. Sham block or contralateral sciatic nerve block did not differ from perineural saline injection throughout the study in either model. The effect of tramadol was not antagonized by intraperitoneal naloxone. High dose tramadol (5 mg) blocked motor function as well as 2% lidocaine. In conclusion, tramadol blocks nociception and motor function in vivo similar to local anesthetics.

Time course of the hemodynamic responses to aortic depressor nerve stimulation in conscious spontaneously hypertensive rats

The time to reach the maximum response of arterial pressure, heart rate and vascular resistance (hindquarter and mesenteric) was measured in conscious male spontaneously hypertensive (SHR) and normotensive control rats (NCR; Wistar; 18-22 weeks) subjected to electrical stimulation of the aortic depressor nerve (ADN) under thiopental anesthesia. The parameters of stimulation were 1 mA intensity and 2 ms pulse length applied for 5 s, using frequencies of 10, 30, and 90 Hz. The time to reach the hemodynamic responses at different frequencies of ADN stimulation was similar for SHR (N = 15) and NCR (N = 14); hypotension = NCR (4194 ± 336 to 3695 ± 463 ms) vs SHR (3475 ± 354 to 4494 ± 300 ms); bradycardia = NCR (1618 ± 152 to 1358 ± 185 ms) vs SHR (1911 ± 323 to 1852 ± 431 ms), and the fall in hindquarter vascular resistance = NCR (6054 ± 486 to 6550 ± 847 ms) vs SHR (4849 ± 918 to 4926 ± 646 ms); mesenteric = NCR (5574 ± 790 to 5752 ± 539 ms) vs SHR (5638 ± 648 to 6777 ± 624 ms). In addition, ADN stimulation produced baroreflex responses characterized by a faster cardiac effect followed by a vascular effect, which together contributed to the decrease in arterial pressure. Therefore, the results indicate that there is no alteration in the conduction of the electrical impulse after the site of baroreceptor mechanical transduction in the baroreflex pathway (central and/or efferent) in conscious SHR compared to NCR.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Electroretinography in dogs using a fiber electrode prototype

We compared two electroretinography (ERG) electrodes in dogs using ERG standards of the International Society for Clinical Electrophysiology of Vision (ISCEV). Ten healthy Yorkshire terrier dogs (mean age, 2.80 ± 1.42 years; 6 females) weighing 5.20 ± 1.56 kg were evaluated using an ERG system for veterinary use. Dark- and light-adapted ERG responses were recorded using an ERG-Jet electrode and a fiber electrode prototype. The examinations were performed during 2 visits, 3 weeks apart. Both electrodes (ERG-Jet or fiber prototype) were used on each animal and the first eye to be recorded (OD × OS) was selected randomly. Three weeks later the examination was repeated on the same animal switching the type of electrode to be used that day and the first eye to be examined. The magnitude and waveform quality obtained with the two electrode types were similar for all ERG responses. ERG amplitudes and implicit times obtained from dogs using the fiber electrode prototype were comparable to those obtained with the ERG-Jet electrode for rod, maximal rod-cone summed, cone, and 30-Hz flicker responses. The fiber electrode prototype is a low-cost device, available as an alternative instrument for clinical veterinary ERG recording for retinal function assessment.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

Roles of estrogen and progesterone in modulating renal nerve function in the rat kidney

The maintenance of extracellular Na+ and Cl- concentrations in mammals depends, at least in part, on renal function. It has been shown that neural and endocrine mechanisms regulate extracellular fluid volume and transport of electrolytes along nephrons. Studies of sex hormones and renal nerves suggested that sex hormones modulate renal function, although this relationship is not well understood in the kidney. To better understand the role of these hormones on the effects that renal nerves have on Na+ and Cl- reabsorption, we studied the effects of renal denervation and oophorectomy in female rats. Oophorectomized (OVX) rats received 17β-estradiol benzoate (OVE, 2.0 mg·kg-1·day-1, sc) and progesterone (OVP, 1.7 mg·kg-1·day-1,sc). We assessed Na+ and Cl-fractional excretion (FENa+ and FECl-, respectively) and renal and plasma catecholamine release concentrations. FENa+, FECl-, water intake, urinary flow, and renal and plasma catecholamine release levels increased in OVX vs control rats. These effects were reversed by 17β-estradiol benzoate but not by progesterone. Renal denervation did not alter FENa+, FECl-, water intake, or urinary flow values vs controls. However, the renal catecholamine release level was decreased in the OVP (236.6±36.1 ng/g) and denervated rat groups (D: 102.1±15.7; ODE: 108.7±23.2; ODP: 101.1±22.1 ng/g). Furthermore, combining OVX + D (OD: 111.9±25.4) decreased renal catecholamine release levels compared to either treatment alone. OVE normalized and OVP reduced renal catecholamine release levels, and the effects on plasma catecholamine release levels were reversed by ODE and ODP replacement in OD. These data suggest that progesterone may influence catecholamine release levels by renal innervation and that there are complex interactions among renal nerves, estrogen, and progesterone in the modulation of renal function.

Limb immobilization alters functional electrophysiological parameters of sciatic nerve

Immobilization, used in clinical practice to treat traumatologic problems, causes changes in muscle, but it is not known whether changes also occur in nerves. We investigated the effects of immobilization on excitability and compound action potential (CAP) and the ultrastructure of the rat sciatic nerve. Fourteen days after immobilization of the right leg of adult male Wistar rats (n=34), animals were killed and the right sciatic nerve was dissected and mounted in a moist chamber. Nerves were stimulated at a baseline frequency of 0.2 Hz and tested for 2 min at 20, 50, and 100 Hz. Immobilization altered nerve excitability. Rheobase and chronaxy changed from 3.13±0.05 V and 52.31±1.95 µs (control group, n=13) to 2.84±0.06 V and 59.71±2.79 µs (immobilized group, n=15), respectively. Immobilization altered the amplitude of CAP waves and decreased the conduction velocity of the first CAP wave (from 93.63±7.49 to 79.14±5.59 m/s) but not of the second wave. Transmission electron microscopy showed fragmentation of the myelin sheath of the sciatic nerve of immobilized limbs and degeneration of the axon. In conclusion, we demonstrated that long-lasting leg immobilization can induce alterations in nerve function.

Effects of different frequencies of transcutaneous electrical nerve stimulation on venous vascular reactivity

Transcutaneous electrical nerve stimulation (TENS) is a type of therapy used primarily for analgesia, but also presents changes in the cardiovascular system responses; its effects are dependent upon application parameters. Alterations to the cardiovascular system suggest that TENS may modify venous vascular response. The objective of this study was to evaluate the effects of TENS at different frequencies (10 and 100 Hz) on venous vascular reactivity in healthy subjects. Twenty-nine healthy male volunteers were randomized into three groups: placebo (n=10), low-frequency TENS (10 Hz, n=9) and high-frequency TENS (100 Hz, n=10). TENS was applied for 30 min in the nervous plexus trajectory from the superior member (from cervical to dorsal region of the fist) at low (10 Hz/200 μs) and high frequency (100 Hz/200 μs) with its intensity adjusted below the motor threshold and intensified every 5 min, intending to avoid accommodation. Venous vascular reactivity in response to phenylephrine, acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) was assessed by the dorsal hand vein technique. The phenylephrine effective dose to achieve 70% vasoconstriction was reduced 53% (P<0.01) using low-frequency TENS (10 Hz), while in high-frequency stimulation (100 Hz), a 47% increased dose was needed (P<0.01). The endothelium-dependent (acetylcholine) and independent (sodium nitroprusside) responses were not modified by TENS, which modifies venous responsiveness, and increases the low-frequency sensitivity of α1-adrenergic receptors and shows high-frequency opposite effects. These changes represent an important vascular effect caused by TENS with implications for hemodynamics, inflammation and analgesia.

Effects of acute administration of selective serotonin reuptake inhibitors on sympathetic nerve activity

Serotonergic mechanisms have an important function in the central control of circulation. Here, the acute effects of three selective serotonin (5-HT) reuptake inhibitors (SSRIs) on autonomic and cardiorespiratory variables were measured in rats. Although SSRIs require 2-3 weeks to achieve their full antidepressant effects, it has been shown that they cause an immediate inhibition of 5-HT reuptake. Seventy male Wistar rats were anesthetized with urethane and instrumented to record blood pressure, heart rate, renal sympathetic nerve activity (RSNA), and respiratory frequency. At lower doses, the acute cardiovascular effects of fluoxetine, paroxetine and sertraline administered intravenously were insignificant and variable. At middle and higher doses, a general pattern was observed, with significant reductions in sympathetic nerve activity. At 10 min, fluoxetine (3 and 10 mg/kg) reduced RSNA by -33±4.7 and -31±5.4%, respectively, without changes in blood pressure; 3 and 10 mg/kg paroxetine reduced RSNA by -35±5.4 and -31±5.5%, respectively, with an increase in blood pressure +26.3±2.5; 3 mg/kg sertraline reduced RSNA by -59.4±8.6%, without changes in blood pressure. Sympathoinhibition began 5 min after injection and lasted approximately 30 min. For fluoxetine and sertraline, but not paroxetine, there was a reduction in heart rate that was nearly parallel to the sympathoinhibition. The effect of these drugs on the other variables was insignificant. In conclusion, acute peripheral administration of SSRIs caused early autonomic cardiovascular effects, particularly sympathoinhibition, as measured by RSNA. Although a peripheral action cannot be ruled out, such effects are presumably mostly central.

Oat hulls treated with alkaline hydrogen peroxide associated with extrusion as fiber source in cookies

Cookies were prepared with the replacement of 20% of wheat flour by chemically (alkaline hydrogen peroxide) and physically (extrusion) treated oat hulls, with the objective to investigate the possibility of use of this modified material. Cookies elaborated with the untreated hulls were used as control. Cookies were evaluated for their physical (spread ratio, specific volume and color) and sensory characteristics, and no difference was detected (p<0.05) among the cookies in relation to the physical properties. Triangule test, used to verify difference (p<0.05) among treated and untreated cookies, confirmed the efficiency of the treatment in sensory level. The acceptance level of cookies with treated fiber was evaluated by potential consumers of the product, obtaining 91% acceptance. The cookies presented 10.6 g of dietary fiber per 100 g of product.