Immune reactivity and immunosuppressive intervention in experimental nephritis. II. Effect of TLI on the course of two models of nephritis in the inbred rat

Fractionated high-dose (3400 rad) total lymphoid irradiation (TLI) induces a unique and prolonged state of immunologic unresponsiveness. The therapeutic efficacy of TLI in immune glomerular disease was explored in two animal models: the accelerated autologous form of nephrotoxic serum nephritis (AA-NTSN) and autologous immune complex nephritis (AICN). LEW rats with established AA-NTSN, subjected to TLI, manifest decreased levels of circulating antibody to the heterologous (sheep) immunoglobulin G (0.4 +/- 0.2 vs 1.2 +/- 0.3 mg/ml, mean +/- SE respectively, p less than 0.01) early post TLI in association with a reduction in histopathology and albuminuria (6.7 +/- 2.2 vs control 19.6 +/- 5.4 mg/24 hr, mean +/- SE, p less than 0.02). Administration of TLI to rats with established AICN effected significant (p less than 0.001) reduction in albuminuria (162 +/- 30 vs 315 +/- 27), serum creatinine (p less than 0.005), and the incidence of lipemia (p less than 0.01) vs controls. Adoptive transfer studies provided no evidence that the sustained beneficial effect of TLI in AICN was suppressor cell mediated. Thus, the observed therapeutic efficacy of TLI in the treatment of experimental nephritis, shown to be related to a reduction in the level of circulating antibody in AA-NTSN, provides a new model system for study of immunity and immunosuppression in primary glomerular disease.     

Species differences in reactivity of mouse and rat astrocytes in vitro

Reactive astrogliosis constitutes a major obstacle to neuronal regeneration and is characterized by rearrangement and upregulation of expression of cytoskeletal proteins, increased proliferation and hypertrophy. Many approaches have been attempted to mimic astrogliosis by inducing reactive astrocytes in vitro. Such research is usually performed using astrocytes derived from Mus musculus or Rattus norvegicus, and results compared between species on the assumption that these cells behave equivalently. Therefore, we compared reactivity between mouse and rat astrocytes in scratch wound assays to gain further insight into how comparable these cell culture models are. Proliferation and migration, as well as expression of the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin, were compared by immunocytochemistry and immunoblot. Further, we investigated migration of proliferating cells by 5-ethynyl-2'-deoxyuridine staining. Substantial differences in GFAP expression and proliferation between astrocytes of the two species were found: rat astrocytes showed different cytoskeletal morphology, expressed significantly more GFAP and vimentin of different molecular size and were more proliferative than comparable mouse astrocytes. Our results suggest that rat and mouse astrocytes may respond differently to various reactivity-triggering stimuli, which needs to be considered when general conclusions are drawn regarding effects of factors regulating astrocyte reactivity.     

Time course of Keap1-Nrf2 pathway expression after experimental intracerebral haemorrhage: correlation with brain oedema and neurological deficit

Abstract

Oxidative stress (OS) is involved in the progression of intracerebral haemorrhage (ICH)-induced secondary brain injury. The pathway involving Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) is currently recognised as the major endogenous regulatory system against oxidative injury. Although its beneficial role has been described for ICH, the time course of Keap1-Nrf2 pathway expression, the activity of downstream antioxidative enzymes, and the association with brain oedema and neurological deficits have not been fully investigated. In this study, we investigated the temporal changes in expression of Keap1, Nrf2, and their downstream antioxidative proteins in the ICH rat brain. We additionally quantified the relationship between these gene and protein changes with brain water content and neurological behaviour scores. After blood infusion, Keap1 showed decreased expression starting at 8 h, whereas Nrf2 began to show a significant increase at 2 h with a peak at 24 h. Keap1 and Nrf2 are chiefly expressed in neuronal cells but not in glial cells. The downstream antioxidative enzymes such as haemeoxygenase-1 (HO-1), glutathione (GSH), thioredoxin (TRX), and glutathione-S-transferase (GST-α1) increased to different degrees during the early stages of ICH. Among these enzymes, HO-1 showed a significant time-dependent increase starting 8 h after ICH. In addition, there was a positive correlation between the HO-1 level and brain water content. In combination, these results suggest that activation of the Keap1-Nrf2 pathway may play an important endogenous neuroprotective role during OS after ICH. Because HO-1 expression is temporally associated with brain oedema – reflective of the severity of brain injury – it may be used as a biomarker of haeme-mediated oxidative damage after ICH.     

The influence of function on the development of bone curvature. An experimental study on the rat tibia

The effect of depriving the tibia of normal mechanical function during its growing period was investigated by performing a unilateral sciatic neurectomy in the gluteal region of young rats. The animals were kept for 16 months, after which their tibias were examined. On the neurectomized side there was a failure of this bone to attain its normal weight, thickness, cross sectional shape or longitudinal curvature. Attainment of the bone's overall length was uneffected.     

Umbilical cord-derived MSC and hyperbaric oxygen therapy effectively protected the brain in rat after acute intracerebral haemorrhage

This study tested the hypothesis that combined therapy with human umbilical cord-derived mesenchymal stem cells (HUCDMSCs) and hyperbaric oxygen (HBO) was superior to either one on preserving neurological function and reducing brain haemorrhagic volume (BHV) in rat after acute intracerebral haemorrhage (ICH) induced by intracranial injection of collagenase. Adult male SD rats (n = 30) were equally divided into group 1 (sham-operated control), group 2 (ICH), group 3 (ICH +HUCDMSCs/1.2 × 10⁶ cells/intravenous injection at 3h and days 1 and 2 after ICH), group 4 (ICH +HBO/at 3 hours and days 1 and 2 after ICH) and group 5 (ICH +HUCDMSCs-HBO), and killed by day 28 after ICH. By day 1, the neurological function was significantly impaired in groups 2-5 than in group 1 (P < .001), but it did not differ among groups 2 to 5. By days 7, 14 and 28, the integrity of neurological function was highest in group 1, lowest in group 2 and significantly progressively improved from groups 3 to 5 (all P < .001). By day 28, the BHV was lowest in group 1, highest in group 2 and significantly lower in group 5 than in groups 3/4 (all P < .0001). The protein expressions of inflammation (HMGB1/TLR-2/TLR-4/MyD88/TRAF6/p-NF-κB/IFN-γ/IL-1ß/TNF-α), oxidative stress/autophagy (NOX-1/NOX-2/oxidized protein/ratio of LC3B-II/LC3B-I) and apoptosis (cleaved-capspase3/PARP), and cellular expressions of inflammation (CD14+, F4/80+) in brain tissues exhibited an identical pattern, whereas cellular levels of angiogenesis (CD31+/vWF+/small-vessel number) and number of neurons (NeuN+) exhibited an opposite pattern of BHV among the groups (all P < .0001). These results indicate that combined HUCDMSC-HBO therapy offered better outcomes after rat ICH.     

Intra-pancreatic release of bradykinin during the course experimental pancreatitis in rat

Kallikrein/Kininogn activation is an important pathophysiological event in acute pancreatitis, leading to microcirculatory changes within the gland. Hitherto, only indirect measurements of pancreatic bradykinin formation have been performed, monitoring the peptide in the circulation and in the peritoneal exudate. In the present study, intra-pancreatic bradykinin release was assessed using microdialysis during experimental acute pancreatitis in rat. In mild, oedematous pancreatitis, induced by caerulein hyperstimulation, the levels of bradykinin within the gland were not elevated compared with those of control rats. However, in necrotic pancreatitis, induced by retrograde injection of taurocholate into the pancreatic duct, significantly elevated levels of intraglandular bradykinin were seen. Several rats in this group died whilst in a state of circulatory shock.     

The influence of chlortetracycline on the immunological reactivity of chickens

Morphological examination of spleen and bursa of Fabricius of chickens fed on a diet without or with the addition of chlortetracycline (50, 100 and 200 CTC mg/kg) has shown that both low and high doses of antibiotic in the diet do not influence significantly the structure and reactivity of the lymphatic tissue following the antigenic stimulation. Certain differences were found in the number of lymphatic follicles in the spleen between control and experimental animals which received the highest dose 200 mg CTC/kg but even the greatest differences found on the fifth day after vaccination were not significant (P<0.1). On the other hand, the study of the level of agglutinin titres in the blood of control and experimental broilers has shown that the doses 100 and 200 mg CTC/kg in the diet have significantly influenced their production. After such doses the latent period was slightly lenghtened and both the highest and the average titres were lowered during the whole postvaccinal period to a degree which correlated the magnitude of dosage of the antibiotic.     

Haematoma scavenging in intracerebral haemorrhage: from mechanisms to the clinic

The products of erythrocyte lyses, haemoglobin (Hb) and haem, are recognized as neurotoxins and the main contributors to delayed cerebral oedema and tissue damage after intracerebral haemorrhage (ICH). Finding a means to efficiently promote absorption of the haemolytic products (Hb and haem) around the bleeding area in the brain through stimulating the function of the body's own garbage cleaning system is a novel clinical challenge and critical for functional recovery after ICH. In this review, available information of the brain injury mechanisms underlying ICH and endogenous haematoma scavenging system is provided. Meanwhile, potential intervention strategies are discussed. Intracerebral blood itself has ‘toxic’ effects beyond its volume effect after ICH. Haptoglobin–Hb–CD163 as well as haemopexin–haem–LRP1 is believed to be the most important endogenous scavenging pathway which participates in blood components resolution following ICH. PPARγ–Nrf2 activates the aforementioned clearance pathway and then accelerates haematoma clearance. Meanwhile, the scavenger receptors as novel targets for therapeutic interventions to treat ICH are also highlighted.     

Expression of Ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat

One question in the pathogenesis of experimental allergic encephalomyelitis (EAE) is whether antigen-presenting cells exist in the central nervous system which help induce the development of the disease. Since EAE is a delayed-type hypersensitivity condition, and since T cells require major histocompatibility complex (MHC)-restricted antigen presentation, it is presumed that if antigen presentation occurs in CNS tissue, the presenting cell should express surface Ia molecules. Using immunofluorescent double labeling, the possibility that astrocytes express surface Ia during EAE evolution in the Lewis rat was examined. Very rare Ia-positive astrocytes were found (less than 0.1% of the astrocytes), but only in the spinal cords of clinically ill animals. In addition, endothelial cell Ia positivity was noted prior to the onset of clinical disease. The immunological significance of such low numbers of astrocytes expressing Ia during EAE is uncertain.     

Xenon induces metaphase arrest in rat astrocytes.

The use of xenon as an almost ideal anesthetic with very little side effects is gaining clinical acceptance, yet its effects on the cellular level are still unclear. It affects intracellular Ca2+-homeostasis but up to now no cellular event or Ca2+-signaling system has been described to be specifically sensitive to xenon. Here we report for the first time a specific effect of xenon on astroglial cells not found with another volatile anesthetic, isoflurane, nor with helium nor with N2: treatment of primary astroglial cells from embryonic rat brain with xenon induces, apart from a slight retardation of the cell cycle, a block at metaphase. Upon removal of xenon cells arrested at metaphase complete their mitosis normally. Even under continuous exposure to xenon, cells can be rescued from metaphase arrest by a small and transient increase in intracellular Ca2+; cells enter anaphase despite the presence of xenon and complete cell division, exhibiting normal rate of chromosome movement and normal chromosome separation. These results suggest that xenon interferes with some Ca2+-dependent regulatory system required for the metaphase-anaphase transition; taking into account its anesthetic effects, xenon may be also involved in the control of glia-mediated signaling transfer.     

The influence of chronic experimental diabetes on contractile responses of rat isolated blood vessels

The influence of experimental diabetes induced by streptozotocin on responses of rat isolated aortae and portal veins to noradrenaline, 5-hydroxytryptamine, and KCl was examined 7, 100, 180, and 360 days after the onset of diabetes. No significant changes in reactivity were seen 7 days after the onset of diabetes. After 100 days aortae from diabetic rats were supersensitive (defined as a significant increase in the pD2 value) to noradrenaline. However, 180 days after the onset of diabetes, the sensitivity of diabetic aortae to noradrenaline was not significantly different from control, while the responsiveness (defined as the maximum developed tension divided by cross-sectional area of aorta) to 5-hydroxytryptamine was reduced. A generalized increase in both the sensitivity and responsiveness of diabetic aortae to all three agonists was observed after 360 days of diabetes. In contrast, no changes in either the sensitivity or the responsiveness of portal veins to noradrenaline, 5-hydroxytryptamine, or KCl could be detected at any time after the onset of diabetes. These results indicate that changes in vascular reactivity can be detected with increasing duration of experimental diabetes. However, these changes do not follow a consistent pattern and are not seen in all parts of the vasculature.     

Action of endothelin-1 on rat astrocytes through the ETB receptor.

We investigated the effect of ET-1 on the state of rat cerebral astrocytes (AC) differentiation. AC ceased to proliferate and changed into its differentiated state by treatment with dibutyryl cyclic AMP (DBcAMP). The cell growth activity in DBcAMP-treated AC was stimulated by ET-1 in a dose-dependent manner. Over similar dose ranges, ET-1 suppressed the glutamine synthetase activity in DBcAMP-treated AC. The molar potency of ET-1 in this action was at least 3 orders of magnitude higher than that in mitogenic action in AC under the proliferative state previously reported. Northern blot analysis revealed that ETB receptor mRNA level in DBcAMP-treated AC was markedly higher than that in AC untreated with DBcAMP. Consistently, binding studies showed that the Bmax value for [125I]ET-1 in DBcAMP-treated AC was 16 times higher than that in AC untreated with DBcAMP. These results suggest that ET-1 potently induced a retraction of the differentiation state of AC from fully the specialized state and that the high responsiveness of differentiated AC to ET-1 was partly attributed to the high level expression of the ETB receptor.