Delayed multidose treatment with nicotinamide extends the degree and duration of neuroprotection by reducing infarction and improving behavioral scores up to two weeks following transient focal cerebral ischemia in Wistar rats.

A single, delayed dose of nicotinamide (NAm) was shown to be protective against focal cerebral ischemia in rats, but the protection was limited to three to seven days following stroke. The investigation reported here was conducted to examine if the use of multiple doses of NAm, administered after the onset of focal cerebral ischemia, would extend the duration of neuroprotection compared with a single dose treatment regimen. Male Wistar rats were subjected to transient focal cerebral ischemia by occluding the right middle cerebral artery (MCAo) for two hours. Following MCAo, motor and sensory behavioral tests were performed daily and the cerebral infarct volumes were measured at two weeks after sacrifice. Each animal was placed into one of four groups that received either normal saline alone (Group S), one (Group A), two (Group B), or three (Group C) doses of NAm (500 mg/kg). Each animal, therefore, received three treatments over two weeks, with the first dose administered intravenously two hours after the onset of MCAo. Single and multiple doses of NAm reduced the infarction (p < 0.01) and improved (p < 0.05) the neurologic sensory and motor behavior when compared with the saline-treated animals up to two weeks after stroke. Moreover, animals that received multiple doses of NAm recuperated full motor function not different from normal, preoperative motor behavior. Delayed treatment with NAm given as multiple doses, therefore, further enhances the extent and duration of neuroprotection by significantly reducing cerebral infarct volumes, improving neurologic behavioral scores, and confers a complete motor recovery up to two weeks from the onset of focal cerebral ischemia in Wistar rats.

Delayed treatment with nicotinamide (Vitamin B(3)) improves neurological outcome and reduces infarct volume after transient focal cerebral ischemia in Wistar rats.

BACKGROUND AND PURPOSE: We have previously shown that nicotinamide (NAm) acutely reduces brain infarction induced by permanent middle cerebral artery occlusion (MCAo) in rats. In this study, we investigate whether NAm may protect against ischemia/reperfusion injury by improving sensory and motor behavior as well as brain infarction volumes in a model of transient focal cerebral ischemia. METHODS: Forty-eight male Wistar rats were used, and transient focal cerebral ischemia was induced by MCAo for 2 hours, followed by reperfusion for either 3 or 7 days. Animals were treated with either intraperitoneal saline or NAm (500 mg/kg) 2 hours after the onset of MCAo (ie, on reperfusion). Sensory and motor behavior scores and body weight were obtained daily, and brain infarction volumes were measured on euthanasia. RESULTS: Relative to treatment with saline, treatment with NAm (500 mg/kg IP) 2 hours after the onset of transient focal cerebral ischemia in Wistar rats significantly improved sensory (38%, P<0.005) and motor (42%, P<0.05) neurological behavior and weight gain (7%, P<0.05) up to 7 days after MCAo. The cerebral infarct volumes were also reduced 46% (P<0.05) at 3 days and 35% (P=0.09) at 7 days after MCAo. CONCLUSIONS: NAm is a robust neuroprotective agent against ischemia/reperfusion-induced brain injury in rats, even when administered up to 2 hours after the onset of stroke. Delayed NAm treatment improved both anatomic and functional indices of brain damage. Further studies are needed to clarify whether multiple doses of NAm will improve the extent and duration of this neuroprotective effect and to determine the mechanism(s) of action underlying the neuroprotection observed. Because NAm is already used clinically in large doses and has few side effects, these results are encouraging for the further examination of the possible use of NAm as a therapeutic neuroprotective agent in the clinical treatment of acute ischemic stroke.

Mexiletine and magnesium independently, but not combined, protect against permanent focal cerebral ischemia in Wistar rats.

The neuroprotective effect of mexiletine (Mex), a potent Na(+) channel blocker which decreases neuronal energy demands and prevents energy depletion during ischemia, was evaluated in Wistar rats subjected to permanent middle cerebral artery (MCA) occlusion. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium (TTC)-stained brain sections. Pretreatment with Mex resulted in a significant infarct volume reduction when administered intraperitoneally, either at the dosage of 50 or 60 mg/kg, 1 hr before MCA occlusion (P < 0.05). Delayed treatment with Mex (50 mg/kg) also had neuroprotective effects when given at 0.5 hr (< 0.05), but not 2-4 hr, after MCA occlusion. Intraarterial administration of MgSO(4) (90 mg/kg), in combination with Mex at 60 mg/kg, showed no additive neuroprotective effect, although each agent independently reduced the MCA occlusion-induced infarction volume (P < 0.05). Our results indicate that a single, acute administration of Mex is neuroprotective against permanent focal cerebral ischemia, but perhaps chronic administration is needed to establish a more effective therapeutic window beyond 0.5 hr. Moreover, the present in vivo data do not favor a combined use of Mg(2+) with Mex for limiting ischemic injury in the brain, since these agents caused cardiopulmonary suppression, which may have led to the loss of the neuroprotective effect of each agent independently.

Nicotinamide reduces infarction up to two hours after the onset of permanent focal cerebral ischemia in Wistar rats.

Ischemia depletes ATP and initiates cascades leading to irreversible tissue injury. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD+) which increases neuronal ATP concentration and protects against malonate-induced neurotoxicity, trauma and nitric oxide toxicity. We therefore examined whether nicotinamide could protect against stroke, using a model of permanent middle cerebral artery occlusion (MCA) occlusion in Wistar rats. Nicotinamide reduced neuronal infarction in a dose-specific manner. Furthermore, nicotinamide (500 mg/kg) reduced infarcts when administered up to 2 h after the onset of permanent MCA occlusion. The mechanism of action underlying the neuroprotection observed with nicotinamide remains to be clarified. These results are potentially important since nicotinamide is already used clinically, though not in the treatment of stroke.

The regulatory role of transforming growth factor-beta in activation of milk mononuclear cells.

PROBLEM: Immunoregulatory role of milk mononuclear cells and cytokines during the involution of the mammary gland. METHOD OF STUDY: Bioassays were used to determine the levels of transforming growth factor beta (TGF-beta) and interleukin-2 (IL-2) and their effects on milk mononuclear cells (MMCs). RESULTS: MMCs collected from involuting glands were less responsive to Con A stimulation than peripheral blood mononuclear cells (PBMCs), and purified huTGF-beta 1 inhibited the activation of both MMCs and PBMCs by Con A. Furthermore, secretions collected over a period of approximately 4 weeks into the involution period contained high levels of active TGF-beta and extremely high levels of latent TGF-beta, MMCs stimulated with Con A produced higher levels of IL-2 than did the PBMCs of the same animals, and bovine milk TGF-beta and huTGF-beta 1 significantly inhibited the IL-2 production by MMCs. Mammary secretions of involuting glands did not contain any detectable levels of IL-2, and they inhibited the growth of the IL-2-dependent bovine lymphoblastoid T cell line (BLTC), even in the presence of 10 U/ml of rBo IL-2. CONCLUSIONS: The findings suggest that TGF-beta is a major immunoregulatory factor in the involuting mammary gland.

Lactation stage-dependent changes of lymphocyte subpopulations in mammary secretions: inversion of CD4+/CD8+ T cell ratios at parturition.

PROBLEM: Determination of lactation stage-dependent changes in levels of lymphocyte subpopulations in milk. METHOD: Flow cytometric assay was used to identify and assay lymphocyte subpopulations in bovine milk at different stages of lactation. RESULTS: Lymphocyte subpopulations in mammary secretions of dairy cows change during the lactation cycle. In involuting glands (dry gland), approximately 80-90% of lymphocytes were CD2+ T cells. The proportion of CD2+ T cells, however, decreased to approximately 50% at the colostral stage an fluctuated between 50 to 60% in normal (mature) milk. Throughout the lactation stages, less than 5% were B cells as identified by the monoclonal antibodies against CD21 and MHC class II antigens. Subset analysis showed, however, that the proportion of CD5+ T cells decreased from 90% in involuting gland secretions to 75% in colostrum (peripartum stage), and to approximately 40-50% in the normal (mature) milk, CD4+ T cells constituted between 45 to 55% of lymphocytes in the dry gland secretion but decreased drastically at parturition and maintained at the level below 20% throughout normal lactation. In contrast, the proportion of CD8+ T cells in the dry gland secretion was low, between 30 to 40%, but increased steadily, in an inversely-related manner with that of CD4+ T cells, to approximately 40-50% at parturition and maintained at approximately 30-40% during the normal lactation stage thereafter. Two-color immunofluorescence study revealed further that practically all of the CD8+ cells in dry gland secretions were CD2+, and approximately 40% of them were CD5-. Throughout the lactation cycle, WC1+ gamma delta T cells comprised only 2 to 5% of lymphocytes in mammary secretions. CONCLUSIONS: T lymphocyte subpopulations change dynamically during stages of the lactation cycle. The selective migration of T lymphocyte subpopulations to and from the mammary gland, and their functional roles in the immune competence and regulation of the dam and sucklings remain to be elucidated.

Age-dependent changes in peripheral blood lymphocyte subpopulations in cattle: a longitudinal study.

Immunofluorescence and flow cytometric analyses were used to study the age-dependent changes in the peripheral blood lymphocyte (PBL) subpopulations in cattle. Four healthy Holstein heifer calves (A, B, C and D), 1-2 months of age, were used in this study. Sequential peripheral blood samples were collected once a month for up to 2-2.5 years, and once at approximately 4 years of age. For the first 6 months of age, the calves had similar proportions of CD2+, CD4+, CD8+ T lymphocytes, CD20+ B lymphocytes and MHC class II+ lymphocytes. From 2 months of age up to 2-2.5 years of age, all animals had similar proportions of CD5+ cells; but during the same period, animals A and B had significantly lower proportions of WC1+ gamma delta T cells than animals C and D. After 7 months of age, however, the proportions of CD2+, CD4+ and CD8+ T cells in PBL of animals A and B significantly decreased, whereas the proportions of both CD20+ B lymphocytes and MHC class II+ lymphocytes significantly increased. In contrast, the proportions of the various PBL subpopulations in animals C and D remained virtually unchanged after 7 months of age. For the first 6 months of age, all the calves showed similar absolute counts of PBL. Thereafter, the absolute counts of PBL in animals A and B significantly increased, but remained virtually unchanged in animals C and D. Throughout the study, from 1-2 months up to 2-2.5 years of age the absolute counts of CD2+, CD4+, CD8+ and WC1+ gamma delta T cells in PBL of the four animals were not significantly different from each other. Up to 6 months of age, the CD4+/ CD8+ ratio in all calves was 2.38 +/- 0.46, but significantly decreased thereafter to 1.81 +/- 0.34. However, there were no significant differences in the CD4+/CD8+ ratios among individual animals. The increase in the absolute counts of PBL in animals A and B, after 7 months of age, was due to an increase in the absolute counts of CD5+ cells, CD20+ B lymphocytes and MHC class II+ lymphocytes. Thus, changes in the percent, but not the absolute counts of T lymphocytes, were due to high percent and absolute counts of B lymphocytes, expressing the CD5 and MHC class II antigens.

Growth regulatory effects of transforming growth factor-beta 1 and interleukin-2 on IL-2 dependent CD4+T lymphoblastoid cell line.

Transforming growth factor-beta 1 (TGF-beta 1) is an immuno-modulatory cytokine which has been shown to modulate the activity of T and B cells. We show here that human TGF-beta 1 inhibited stationary cultures of IL-2 dependent CD4+ bovine lymphoblastoid T cells (BLTC) by down-regulating their IL-2 receptor (IL-2R) expression, arresting cells in the G0/G1 compartment of the cell cycle, and inducing these cells to undergo apoptosis. These events were reversed by the addition of a minimal concentration of IL-2 (2U/ml). In the presence of exogenous IL-2, TGF-beta 1 was found to augment the proliferative response of BLTC through up-regulation of IL-2R expression, allow progression of normal cell cycle, and significantly prevent apoptosis. Our data clearly show that IL-2 and TGF-beta 1, when present alone, have contrasting effects on BLTC. TGF-beta 1 down regulates events that are associated with IL-2 mediated signal. But when present together, IL-2 and TGF-beta 1 upregulate activation signals and proliferation of rapidly dividing CD4+T cells.

Development and characterization of an IL-2-dependent bovine CD4+ lymphoblastoid T-cell line.

A bovine IL-2-dependent lymphoblastoid T-cell line (BLTC) was developed and established from peripheral blood mononuclear cells stimulated with Con A, and subpassaged in conditioned media prepared from Con A-activated bovine lymph node cells. The BLTC cell line is IL-2 dependent, requiring exogenous IL-2 either in the form of recombinant bovine or human IL-2 or conditioned media prepared from Con A-stimulated bovine lymph node cells. Conditioned media prepared from mononuclear cells from other species, such as dogs and rats, were without any stimulatory effects. The growth of these cells was lectin-independent, and monokines such as IL-1 beta and TNF-alpha, or conditioned media from LPS-stimulated macrophases failed to support their growth. Immunophenotyping showed that over 90% of the cells were CD2+, CD3+, and CD4+, 25% were CD5%, less than 2% were CD8+, and less than 2% were CD20+. The cells were negative for the gamma delta 215/300 kDa T-cell markers and the 75/110 kDa monocyte markers. The growth of these cells was inhibited by dibutyryl cAMP in the presence or absence of IL-2, and stimulated by the phorbol ester, PMA, in the absence but not in the presence of IL-2. The effects of IL-2 and PMA were blocked by the protein kinase C inhibitor, H-7, suggesting that IL-2 signaling is closely associated with the PKC activation pathway.