Ultrastructural characterization of bovine umbilical cord blood cells

The umbilical cord blood (UCB) is an important source of pluripotent stem cells, which motivated researches on ontogeny and transplantation. The morphological characterization of umbilical cord cells is the first step to establish subsequent experiments on these areas. Although some information on humans can be found, no data on UCB is available for bovines. Therefore, this work is the first attempt to conduct an ultrastructural characterization of bovine umbilical cord blood. Blood was collected from the umbilical cord of twenty fetuses by punction of the umbilical vein. Samples were processed for whole leucocytes observation by centrifugation and the buffy coat was collected. Cells were washed and pelleted and prepared according to the standard protocol of the transmission electron microscopy. The presence of cells with morphologic characteristics compatible with the precursors from the erythrocytic, neutrophilic, eosinophilic, basophilic, and lymphocytic lineages was observed. Atypical cells with peculiar morphological features, strongly similar to apoptotic cells, were seen. Bovine neutrophils with three types of cytoplasmic granules were also found in the blood. The ultrastructural characteristics of observed bovine UCB cells where similar to those found in other species, suggesting that bovines could possibly constitute an experimental model for approaches on UCB cells research.

Spinal cord compression in cattle after the use of an oily vaccine

An outbreak of compressive myelopathy in cattle associated with the improper use of an oil vaccine is described. Neurological signs were observed in 25 out of 3,000 cattle after 60 days of being vaccinated against foot and mouth disease. The clinical picture was characterized by progressive paralysis of the hind limbs, difficulty in standing up, and sternal recumbency during the course of 2-5 months. A filling defect between the L1 and L3 vertebrae was seen through myelography performed in one of the affected animals. A yellow-gray, granular and irregular mass was observed in four necropsied animals involving the spinal nerve roots and epidural space of the lumbar (L1-L4) spinal cord; the mass was associated with a whitish oily fluid. This fluid was also found in association with necrosis of the longissimus dorsi muscle. Microscopic changes in the epidural space, nerve roots, and spinal musculature were similar and consisted of granulomas or pyogranulomas around circular unstained spaces (vacuoles). These spaces were located between areas of severe diffuse hyaline necrosis of muscle fibers and resembled the drops of oil present in the vaccine.

Morphology and topographic anatomy of the spinal cord of the red-footed tortoise (Geochelone carbonaria Spix, 1824)

The aim of this study was to describe the topography of the spinal cord of the red-footed tortoise to establish a morphological basis for applied research in anesthesiology and morphology. Six tortoises from the state of Maranhão (Brazil) that had died of natural causes were used. The common carotid artery was used to perfuse the arterial system with saline solution (heated to 37ºC) and to fix the material with a 20% formaldehyde solution. The specimens were then placed in a modified decalcifying solution for 72 hours to allow dorsal opening of the carapace with a chisel and an orthopedic hammer. Dissection of the dorsal musculature and sectioning of the vertebral arches were performed to access the spinal cord. The results revealed the spinal cord of G. carbonaria to be an elongated, whitish mass that reached the articulation between the penultimate and last caudal vertebrae. The cervical intumescence (Intumescentia cervicalis) was located between vertebral segments C5 and T1, whereas the lumbosacral intumescence (Intumescentia lumbalis) was located between T6 and Ca1.

Transitional cell carcinoma of urinary bladder with metastasis in lumbar vertebrae and spinal cord compression in an ocelot(Leopardus pardalis)

This paper reports a case of nonpapillary and infiltrative transitional cell carcinoma (TCC) of the urinary bladder with metastasis of lumbar vertebrae and spinal cord compression in an adult female ocelot (Leopardus pardalis), from the Mato Grosso state, Brazil. The ocelot had pelvic limb paralysis and skin ulcers in the posterior region of the body and was submitted to euthanasia procedure. At necropsy was observed a multilobulated and irregular shaped, yellowish to white nodule in the urinary bladder. The nodule had a soft consistency and arised from the mucosa of the urinary bladder extending throughout the muscular layers and the serosa. Nodules of similar appearance infiltrating the vertebral column the at L6 and L7 vertebrae with corresponding spinal canal invasion were also observed. The histological evaluation showed epithelial neoplastic proliferation in the urinary bladder with characteristics of nonpapillary and infiltrative TCC, with positive immunohistochemical staining for pancytokeratin, and strong immunostaining for cytokeratin of low molecular weight, and weak or absent labeling for high molecular weight cytokeratin. This is the first report of TCC of urinary bladder in ocelot in Brazil.

Baroreflex and chemoreflex dysfunction in streptozotocin-diabetic rats

Several investigators have demonstrated that streptozotocin (STZ) diabetes induces changes in the autonomic control of the cardiovascular system. Changes in cardiovascular function may be related to peripheral neuropathy. The aim of the present study was to analyze changes in heart rate (HR) and arterial pressure (AP) as well as baroreflex and chemoreflex sensitivity in STZ-induced diabetic male Wistar rats (STZ, 50 mg/kg, iv, 15 days). Intra-arterial blood pressure signals were obtained for control and diabetic rats (N = 9, each group). Data were processed in a data acquisition system (CODAS, 1 kHz). Baroreflex sensitivity was evaluated by measuring heart rate changes induced by arterial pressure variation produced by phenylephrine and sodium nitroprusside injection. Increasing doses of potassium cyanide (KCN) were used to evaluate bradycardic and pressor responses evoked by chemoreflex activation. STZ induced hyperglycemia (447 ± 49 vs 126 ± 3 mg/dl), and a reduction in AP (99 ± 3 vs 118 ± 2 mmHg), resting HR (296 ± 11 vs 355 ± 16 bpm) and plasma insulin levels (16 ± 1 vs 57 ± 11 µU/ml). We also observed that the reflex bradycardia (-1.68 ± 0.1 vs -1.25 ± 0.1 bpm/mmHg, in the diabetic group) and tachycardia (-3.68 ± 0.5 vs -1.75 ± 0.3 bpm/mmHg, in the diabetic group) produced by vasopressor and depressor agents were impaired in the diabetic group. Bradycardia evoked by chemoreflex activation was attenuated in diabetic rats (control: -17 ± 1, -86 ± 19, -185 ± 18, -208 ± 17 vs diabetic: -7 ± 1, -23 ± 5, -95 ± 13, -140 ± 13 bpm), as also was the pressor response (control: 6 ± 1, 30 ± 7, 54 ± 4, 59 ± 5 vs diabetic: 6 ± 1, 8 ± 2, 33 ± 4, 42 ± 5 mmHg). In conclusion, the cardiovascular responses evoked by baroreflex and chemoreflex activation are impaired in diabetic rats. The alterations of cardiovascular responses may be secondary to the autonomic dysfunction of cardiovascular control

Paraquat poisoning: an experimental model of dose-dependent acute lung injury due to surfactant dysfunction

Since the most characteristic feature of paraquat poisoning is lung damage, a prospective controlled study was performed on excised rat lungs in order to estimate the intensity of lesion after different doses. Twenty-five male, 2-3-month-old non-SPF Wistar rats, divided into 5 groups, received paraquat dichloride in a single intraperitoneal injection (0, 1, 5, 25, or 50 mg/kg body weight) 24 h before the experiment. Static pressure-volume (PV) curves were performed in air- and saline-filled lungs; an estimator of surface tension and tissue works was computed by integrating the area of both curves and reported as work/ml of volume displacement. Paraquat induced a dose-dependent increase of inspiratory surface tension work that reached a significant two-fold order of magnitude for 25 and 50 mg/kg body weight (P<0.05, ANOVA), sparing lung tissue. This kind of lesion was probably due to functional abnormalities of the surfactant system, as was shown by the increase in the hysteresis of the paraquat groups at the highest doses. Hence, paraquat poisoning provides a suitable model of acute lung injury with alveolar instability that can be easily used in experimental protocols of mechanical ventilation

Inflammatory response of the spinal cord to multiple episodes of blood-brain barrier disruption and toxic demyelination in Wistar rats

Multiple episodes of blood-brain barrier disruption were induced by sequential intraspinal injections of ethidium bromide. In addition to the barrier disruption, there was toxic demyelination and exposure of myelin components to the immune system. Twenty-seven 3-month-old Wistar rats received 2, 3 or 4 injections of 1 µl of either 0.1% ethidium bromide in normal saline (19 rats) or 0.9% saline (8 rats) at different levels of the spinal cord. The time intervals between the injections ranged from 28 to 42 days. Ten days after the last injection, all rats were perfused with 2.5% glutaraldehyde. The spinal sections were evaluated macroscopically and by light and transmission electron microscopy. All the lesions demonstrated a mononuclear phagocytic infiltrate apparently removing myelin. Lymphocytes were not conspicuous and were found in only 34% of the lesions. No perivascular cuffings were detected. In older lesions (38 days and older) they were found only within Virchow-Robin spaces. This result suggests that multiple blood-brain barrier disruptions with demyelination and exposure of myelin components to the immune system were not sufficient to induce an immune-mediated reaction in the central nervous system.

Renal dysfunction in patients with sickle cell anemia or sickle cell trait

Patients with sickle cell anemia (Hb SS) or sickle cell trait (Hb AS) may present several types of renal dysfunction; however, comparison of the prevalence of these abnormalities between these two groups and correlation with the duration of disease in a large number of patients have not been thoroughly investigated. In a cross-sectional study using immunoenzymometric assays to measure tubular proteinuria, microalbuminuria, measurement of creatinine clearance, urinary osmolality and analysis of urine sediment, we evaluated glomerular and tubular renal function in 106 adults and children with Hb SS (N = 66) or Hb AS (N = 40) with no renal failure (glomerular filtration rate (GFR) >85 ml/min). The percentage of individuals with microalbuminuria was higher among Hb SS than among Hb AS patients (30 vs 8%, P<0.0001). The prevalence of microhematuria was similar in both groups (26 vs 30%, respectively). Increased urinary levels of retinol-binding protein or ß2-microglobulin were detected in only 3 Hb SS and 2 Hb AS patients. Urinary osmolality was reduced in patients with Hb SS or with Hb AS; however, it was particularly evident in Hb SS patients older than 15 years (median = 393 mOsm/kg, range = 366-469) compared with Hb AS patients (median = 541 mOsm/kg, range = 406-722). Thus, in addition to the frequently reported early reduction of urinary osmolality and increased GFR, nondysmorphic hematuria was found in 26 and 30% of patients with Hb SS or Hb AS, respectively. Microalbuminuria is an important marker of glomerular injury in patients with Hb SS and may also be demonstrated in some Hb AS individuals. Significant proximal tubular dysfunction is not a common feature in Hb SS and Hb AS population at this stage of the disease (i.e., GFR >85 ml/min).

Identification of the main generator source of longitudinal muscle contraction in the earthworm ventral nerve cord

The main generator source of a longitudinal muscle contraction was identified as an M (mechanical-stimulus-sensitive) circuit composed of a presynaptic M-1 neuron and a postsynaptic M-2 neuron in the ventral nerve cord of the earthworm, Amynthas hawayanus, by simultaneous intracellular response recording and Lucifer Yellow-CH injection with two microelectrodes. Five-peaked responses were evoked in both neurons by a mechanical, but not by an electrical, stimulus to the mechanoreceptor in the shaft of a seta at the opposite side of an epidermis-muscle-nerve-cord preparation. This response was correlated to 84% of the amplitude, 73% of the rising rate and 81% of the duration of a longitudinal muscle contraction recorded by a mechano-electrical transducer after eliminating the other possible generator sources by partitioning the epidermis-muscle piece of this preparation. The pre- and postsynaptic relationship between these two neurons was determined by alternately stimulating and recording with two microelectrodes. Images of the Lucifer Yellow-CH-filled M-1 and M-2 neurons showed that both of them are composed of bundles of longitudinal processes situated on the side of the nerve cord opposite to stimulation. The M-1 neuron has an afferent process (A1) in the first nerve at the stimulated side of this preparation and the M-2 neuron has two efferent processes (E1 and E3) in the first and third nerves at the recording side where their effector muscle cell was identified by a third microelectrode.

Decreased gastric emptying and gastrointestinal and intestinal transits of liquid after complete spinal cord transection in awake rats

We studied the effect of complete spinal cord transection (SCT) on gastric emptying (GE) and on gastrointestinal (GI) and intestinal transits of liquid in awake rats using the phenol red method. Male Wistar rats (N = 65) weighing 180-200 g were fasted for 24 h and complete SCT was performed between C7 and T1 vertebrae after a careful midline dorsal incision. GE and GI and intestinal transits were measured 15 min, 6 h or 24 h after recovery from anesthesia. A test meal (0.5 mg/ml phenol red in 5% glucose solution) was administered intragastrically (1.5 ml) and the animals were sacrificed by an iv thiopental overdose 10 min later to evaluate GE and GI transit. For intestinal transit measurements, 1 ml of the test meal was administered into the proximal duodenum through a cannula inserted into a gastric fistula. GE was inhibited (P<0.05) by 34.3, 23.4 and 22.7%, respectively, at 15 min, 6 h and 24 h after SCT. GI transit was inhibited (P<0.05) by 42.5, 19.8 and 18.4%, respectively, at 15 min, 6 h and 24 h after SCT. Intestinal transit was also inhibited (P<0.05) by 48.8, 47.2 and 40.1%, respectively, at 15 min, 6 h and 24 h after SCT. Mean arterial pressure was significantly decreased (P<0.05) by 48.5, 46.8 and 41.5%, respectively, at 15 min, 6 h and 24 h after SCT. In summary, our report describes a decreased GE and GI and intestinal transits in awake rats within the first 24 h after high SCT.

Axotomy increases NADPH-diaphorase activity in the dorsal root ganglia and lumbar spinal cord of the turtle Trachemys dorbigni

Seven days after transection of the sciatic nerve NADPH-diaphorase activity increased in the small and medium neurons of the dorsal root ganglia of the turtle. However, this increase was observed only in medium neurons for up to 90 days. At this time a bilateral increase of NADPH-diaphorase staining was observed in all areas and neuronal types of the dorsal horn, and in positive motoneurons in the lumbar spinal cord, ipsilateral to the lesion. A similar increase was also demonstrable in spinal glial and endothelial cells. These findings are discussed in relation to the role of nitric oxide in hyperalgesia and neuronal regeneration or degeneration.

Induction of Fos protein immunoreactivity by spinal cord contusion

The objective of the present study was to identify neurons in the central nervous system that respond to spinal contusion injury in the rat by monitoring the expression of the nuclear protein encoded by the c-fos gene, an activity-dependent gene, in spinal cord and brainstem regions. Rats were anesthetized with urethane and the injury was produced by dropping a 5-g weight from 20.0 cm onto the exposed dura at the T10-L1 vertebral level (contusion group). The spinal cord was exposed but not lesioned in anesthetized control animals (laminectomy group); intact animals were also subjected to anesthesia (intact control). Behavioral alterations were analyzed by Tarlov/Bohlman scores, 2 h after the procedures and the animals were then perfused for immunocytochemistry. The patterns of Fos-like immunoreactivity (FLI) which were site-specific, reproducible and correlated with spinal laminae that respond predominantly to noxious stimulation or injury: laminae I-II (outer substantia gelatinosa) and X and the nucleus of the intermediolateral cell column. At the brain stem level FLI was detected in the reticular formation, area postrema and solitary tract nucleus of lesioned animals. No Fos staining was detected by immunocytochemistry in the intact control group. However, detection of FLI in the group submitted to anesthesia and surgical procedures, although less intense than in the lesion group, indicated that microtraumas may occur which are not detected by the Tarlov/Bohlman scores. There is both a local and remote effect of a distal contusion on the spinal cord of rats, implicating sensory neurons and centers related to autonomic control in the reaction to this kind of injury.

Effects of exercise training on autonomic and myocardial dysfunction in streptozotocin-diabetic rats

Several investigators have demonstrated that diabetes is associated with autonomic and myocardial dysfunction. Exercise training is an efficient non-pharmacological treatment for cardiac and metabolic diseases. The aim of the present study was to investigate the effects of exercise training on hemodynamic and autonomic diabetic dysfunction. After 1 week of diabetes induction (streptozotocin, 50 mg/kg, iv), male Wistar rats (222 ± 5 g, N = 18) were submitted to exercise training for 10 weeks on a treadmill. Arterial pressure signals were obtained and processed with a data acquisition system. Autonomic function and intrinsic heart rate were studied by injecting methylatropine and propranolol. Left ventricular function was assessed in hearts perfused in vitro by the Langendorff technique. Diabetes (D) bradycardia and hypotension (D: 279 ± 9 bpm and 91 ± 4 mmHg vs 315 ± 11 bpm and 111 ± 4 mmHg in controls, C) were attenuated by training (TD: 305 ± 7 bpm and 100 ± 4 mmHg). Vagal tonus was decreased in the diabetic groups and sympathetic tonus was similar in all animals. Intrinsic heart rate was lower in D (284 ± 11 bpm) compared to C and TD (390 ± 8 and 342 ± 14 bpm, respectively). Peak systolic pressure developed at different pressures was similar for all groups, but +dP/dt max was decreased and -dP/dt max was increased in D. In conclusion, exercise training reversed hypotension and bradycardia and improved myocardial function in diabetic rats. These changes represent an adaptive response to the demands of training, supporting a positive role of physical activity in the management of diabetes.

Coronary endothelial dysfunction after ischemia and reperfusion: a new therapeutic target?

Although cardiac ischemia is usually characterized as a disease of the myocyte, it is clear that the vasculature, and especially endothelial cells, is also a major target of this pathology. Indeed, using a rat model of ischemia/reperfusion, we were able to detect severe endothelial dysfunction (assessed as a decreased response to acetylcholine) after acute or chronic reperfusion. Given the essential role of the endothelium in the regulation of vascular tone, as well as platelet and leukocyte function, such a severe dysfunction could lead to an increased risk of vasospasm, thrombosis and accelerated atherosclerosis. This dysfunction can be prevented by free radical scavengers and by exogenous nitric oxide. Endothelial dysfunction can also be prevented by preconditioning with brief periods of intermittent ischemia, thus extending to coronary endothelial cells the concept of endogenous protection previously described at the myocyte level. Experiments performed on cultured cells showed that the endothelial protection induced by free radical scavengers or by preconditioning was due to a lesser expression of endothelial adhesion molecules such as intercellular adhesion molecule-1, leading to a lesser adhesion of neutrophils to endothelial cells. Identification of the mechanisms of this protection may lead to the development of new strategies aimed at protecting the vasculature in ischemic heart diseases.

Detection of renal dysfunction based on serum creatinine levels in a Brazilian community: the Bambuí Health and Ageing Study

There are few population-based studies of renal dysfunction and none conducted in developing countries. In the present study the prevalence and predictors of elevated serum creatinine levels (SCr > or = 1.3 mg/dl for men and 1.1 mg/dl for women) were determined among Brazilian adults (18-59 years) and older adults (>60 years). Participants included all older adults (N = 1742) and a probabilistic sample of adults (N = 818) from Bambuí town, MG, Southeast Brazil. Predictors were investigated using multiple logistic regression. Mean SCr levels were 0.77 ± 0.15 mg/dl for adults, 1.02 ± 0.39 mg/dl for older men, and 0.81 ± 0.17 mg/dl for older women. Because there were only 4 cases (0.48%) with elevated SCr levels among adults, the analysis of elevated SCr levels was restricted to older adults. The overall prevalence of elevated SCr levels among the elderly was 5.09% (76/1494). The prevalence of hypercreatinemia increased significantly with age (chi² = 26.17, P = 0.000), being higher for older men (8.19%) than for older women (5.29%, chi² = 5.00, P = 0.02). Elevated SCr levels were associated with age 70-79 years (odds ratio [OR] = 2.25, 95% confidence interval [CI]: 1.15-4.42), hypertension (OR = 3.04, 95% CI: 1.34-6.92), use of antihypertensive drugs (OR = 2.46, 95% CI: 1.26-4.82), chest pain (OR = 3.37, 95% CI: 1.31-8.74), and claudication (OR = 3.43, 95% CI: 1.30-9.09) among men, and with age >80 years (OR = 4.88, 95% CI: 2.24-10.65), use of antihypertensive drugs (OR = 4.06, 95% CI: 1.67-9.86), physical inactivity (OR = 2.11, 95% CI: 1.11-4.02) and myocardial infarction (OR = 3.89, 95% CI: 1.58-9.62) among women. The prevalence of renal dysfunction observed was much lower than that reported in other population-based studies, but predictors were similar. New investigations are needed to confirm the variability in prevalence and associated factors of renal dysfunction among populations.