Differential upregulation of heme oxygenase-1 (HSP32) in glial cells after oxidative stress and in demyelinating disorders

Oxidative stress is implicated in the pathogenesis of demyelinating disorders and inflammatory responses. Heme oxygenase-1 (HO-1; HSP32) is a small heat shock protein (HSP) with enzymatic activity, which is inducible by oxidative stress. In this study we analyzed autopsy and biopsy brain samples of patients with multiple sclerosis (MS) and ADEM (acute disseminated leucoencephalomyelits) and spinal cord lesions of mouse EAE (experimental autoimmune encephalomyelitis), which was actively induced by immunization with myelin oligodendrocyte glycoprotein (MOG_35–55) peptide, for the presence of HO-1. HO-1 was observed in glial cells during different stages: (1) during acute phases of mainly inflammatory diseases (EAE and ADEM) expression of HO-1 was prominent in microglia/macrophages and astrocytes, and upregulation correlated with inflammation, and (2) in early MS lesions HO-1 was expressed in oligodendrocytes. Furthermore, in glial cell cultures, we can show that upregulation of HO-1 in oligodendrocytes was paralleled by severe morphological damage. Oligodendrocytes underwent apoptotic cell death at a concentration of hydrogen peroxide (50–200 μM) which did not affect astrocytes or microglia. Using oligodendroglial OLN-93 cells, we demonstrate that oxidative stress led to mitochondrial impairment and the disorganization of the microtubule network. Zinc protoporphyrin, an inhibitor of HO-1, augmented the cytotoxic consequences of hydrogen peroxide in OLN-93 cells. Hence, the presence of HO-1 in EAE, ADEM, and MS points to the involvement of oxidative stress and a role of HO-1 in the pathogenesis of the diseases. The data suggest that stress-induced HO-1 initially plays a protective role, while its chronic upregulation, might contribute to oligodendroglial cell death rather than providing protection.     

多发性硬化患者抗髓鞘少突胶质细胞糖蛋白抗体的检测及其意义

目的　检测多发性硬化 (MS)患者抗髓鞘少突胶质细胞糖蛋白抗体并探讨其意义。方法　采用EL ISA方法测定 5 6例多发性硬化患者急性期血清和脑脊液配对标本的抗髓鞘少突胶质细胞糖蛋白 (MOG)抗体 ,30例其它神经疾病 (OND)患者为对照。结果　 MS患者血清和脑脊液均可测到抗 MOG抗体 ,血清抗 MOG抗体阳性率为 35 .7% ,脑脊液抗 MOG抗体阳性率为 4 2 .8% ,OND组血清和脑脊液抗 MOG抗体阳性率均为 6 .7% ,与 OND组比较均有显著性意义 (P<0 .0 5 ) ,但血清和脑脊液比较无显著性意义 (P>0 .0 5 )。结论　多发性硬化患者血清和脑脊液中均可检测到抗 MOG抗体 ,可为临床诊断和治疗提供指导。     

Changes in cannabinoid CB(1) receptors in striatal and cortical regions of rats with experimental allergic encephalomyelitis, an animal model of multiple sclerosis

Data, initially anecdotal, but recently supported on more solid experimental evidence, suggest that cannabinoids might be beneficial in the treatment of some of the symptoms of multiple sclerosis (MS). Despite this evidence, there are no data on the possible changes in cannabinoid CB(1) or CB(2) receptors, the main molecular targets for the action of cannabinoids, either in the postmortem brain of patients with MS or in animal models of this disease. The present study addressed this question using the model of experimental allergic encephalomyelitis (EAE) in Lewis rats generated by inoculation of guinea pig myelin basic protein in Freund's adjuvant. After inoculation, animals were examined daily to detect the appearance of neurological signs. The first signs appeared around day 10 after inoculation, reaching the highest degree by day 13, when animals were sacrificed and their brains removed and used for analysis of CB(1) receptor binding, mRNA levels, and activation of GTP-binding proteins. CB(1) receptor binding and mRNA levels were not affected in EAE rats in brain areas such as the hippocampus, limbic structures, and cerebellum. However, there was a marked decrease in both parameters in the caudate-putamen, both in the lateral and medial parts, although this decrease did not correspond with decreases in binding in the nuclei recipient of striatal output neurons, which suggests that changes in CB(1) receptors are exclusively located in the cell bodies of striatal neurons. In addition, CB(1) receptor binding, but not mRNA levels, also decreased in the cerebral cortex, both in the deep and the superficial layers. The analysis of [(35)S]GTPgammaS binding after activation of CB(1) receptors with WIN55,212-2, a synthetic agonist, revealed that, despite the decrease in the number of CB(1) receptors in EAE rats, these were more efficiently coupled to GTP-binding protein-mediated signaling mechanisms in both the caudate-putamen and the cerebral cortex of these animals. In summary, these data suggest that the generation of EAE in Lewis rats would be associated with changes in CB(1) receptors in striatal and cortical neurons, which might be related to the alleviation of some motor signs observed after the treatment with cannabinoid receptor agonists in similar models of MS in rodents.     

Notch1 and its ligand Jagged1 are present in remyelination in a T-cell- and antibody-mediated model of inflammatory demyelination

The Notch receptor and its ligands are involved in myelination in central nervous system (CNS) development. Re-expression of this pathway in the adult CNS has been proposed to hamper remyelination in multiple sclerosis. Previous studies also revealed that pharmacological inhibition of Notch signaling ameliorates experimental autoimmune encephalomyelitis (EAE). However, in a recent study in toxin-induced demyelination constituents of the Notch signaling pathway were demonstrated in remyelinating lesions indicating that remyelination may occur in the presence of Notch signaling. We examined the expression of Notch1-immunoreactivity (IR) and Jagged1-IR in EAE induced by myelin-oligodendrocyte glycoprotein (MOG). In this model, the combined action of T cells, antibodies and the complement cascade yields a pathology closely reflecting multiple sclerosis. Notch1 and its ligand Jagged1 were differentially expressed in the lesions of MOG-EAE. Notch1-IR on macrophages was highest in actively demyelinating and lowest in remyelinating lesions. The amount of Notch1-positive astrocytes increased during the lesion evolution from demyelination to remyelination. Notch1-positive oligodendrocytes were exclusively present in remyelinating lesions and not found in lesions without signs of remyelination. Astrocytes represented the major source of Jagged1-IR in demyelination and remyelination. In conclusion, our study proves that constituents of the Notch pathway are expressed in remyelination in an animal model of T-cell- and antibody-mediated CNS demyelination. Thus, it is unlikely, at least in the paradigm of MOG-EAE, that Notch signaling is responsible for a failure of remyelination. M. K. S. receives grant support from Biogen Idec; R. W. holds a Heisenberg fellowship of Deutsche Forschungsgemeinschaft (DFG We 1947/4-2).     

Sensorimotor and cognitive function of a NEFL(P22S) mutant model of Charcot-Marie-Tooth disease type 2E

Charcot-Marie-Tooth (CMT) disease is the most frequently encountered hereditary disease causing sensorimotor neuropathies and slowly progressive muscle weakness and atrophy. The P22S mutation of the NEFL gene encoding the light polypeptide neurofilament (NFL) is associated with CMT. To understand more clearly the pathogenesis of sensorimotor dysfunction in CMT, we generated transgenic mice with the NEFL(P22S) mutation under the tet-off tetracycline regulated system with involvement of the Thy1 neuron-specific promoter. NEFL(P22S) transgenic mice exhibited extended duration of the hindlimb clasping response and gait anomalies, as well as sensorimotor deficits in stationary beam and suspended bar tests. In addition, the NEFL(P22S) mice were deficient in the reversal phase of left-right discrimination learning in a water maze. This model mimics some aspects of human CMT pathology and provides an opportunity of ameliorating CMT symptoms with experimental therapies.     

脊髓与背根神经节细胞外信号调节蛋白激酶5(ERK5)信号通路在神经病理性疼痛中的作用的大鼠模型实验

目的 探讨脊髓与背根神经节(DGR)细胞外信号调节蛋白激酶5(ERK5)信号通路在神经病理性疼痛中的作用。方法 选取SD大鼠通过坐骨神经结扎(CCI)建立神经病理性疼痛模型,对建模1、3、6 d脊髓与背根神经节p-ERK5进行免疫组化染色,分析其表达水平变化情况; 利用反义寡核苷酸技术并结合免疫印迹和免疫组化检测DGR中ERK5和p-CREB表达水平的变化; 分析鞘内注射反义寡核苷酸对CCI 大鼠机械缩足反射阈值(MWT)和热缩足反射潜伏期(TWL)的影响。结果 SD大鼠神经病理性疼痛模型建立后p-ERK5阳性神经节数量显著增加; 鞘内注射ERK5反义寡核苷酸可有效抑制脊髓与背根神经节ERK5的表达,同时可上调p-CREB的表达; 大鼠机械缩足反射阈值(MWT)和热缩足反射潜伏期(TWL)均明显下降。结论 脊髓与背根神经节ERK5可能在神经病理性疼痛过程中具有重要调节作用,并且ERK5通过CREB相关基因的表达来发挥其部分功能     

A comparison of clinical and evoked potential (VEP and median nerve SEP) evolution in patients with MS and potentially related conditions

A clinical and electrophysiological (VEP and median nerve SEP) follow-up was performed in 27 subjects selected among 200 consecutive patients previously examined by multimodality evoked potentials because of MS or potentially related conditions. The mean interval between the examinations was 20 months. There was a concordance between the clinical and electrophysiological evolution for the majority of examined pathways. However, alterations were more frequent for evoked potentials than for clinical symptomatology: six VEPs and 16 SEPs were changed compared to the initial recordings whereas clinical alterations occurred in one eye and 11 arms. Definitely contradictory evolutions were observed in only two arms (in one of the MS patients). Significant deteriorations between examinations occurred for two VEPs and two SEPs, although the recordings remained within normal limits. We conclude that evoked potential recordings are valuable for monitoring and that serial recordings may increase the number of abnormal findings.     

Stereoselective effect of (R)- and (S)-1-methyl-1,2,3,4-tetrahydroisoquinolines on a mouse model of Parkinson's disease

We carried out behavioral, pathological, and biochemical studies in order to determine whether the stereo-structure of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) affects the onset of Parkinson's disease-like symptoms, which are induced by 1,2,3,4-tetrahydroisoquinoline (TIQ) in mice. Pretreatment with (R)-1-MeTIQ or its racemate (RS)-1-MeTIQ prevented the TIQ-induced bradykinesia. Pretreatment with a combination of L-DOPA and carbidopa significantly prevented subsequent TIQ-induced bradykinesia. Furthermore, the pathological study demonstrated that either (R)-1-MeTIQ or its racemate protected against TIQ-induced loss of tyrosine hydroxylase-positive cells of the substantia nigra pars compacta. (R)-1-MeTIQ and its racemate also prevented the TIQ-induced reduction in the levels of dopamine and its metabolites in the striatum. Serotonin and its metabolite were not affected by repeated administration of (RS)-1-MeTIQ or its derivatives. On the other hand, (S)-1-MeTIQ induced moderate but significant bradykinesia, whereas (R)-1-MeTIQ did not induce this behavioral abnormality at all. In addition, (S)-enantiomer prevented the onset of TIQ-induced bradykinesia, though to a lesser extent than did either (R)-enantiomer or its racemate. However, (S)-enantiomer did not prevent the loss of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. We concluded that (R)-1-MeTIQ, and not (S)-enantiomer, plays a crucial role in protection against TIQ-induced parkinsonism, a fact which suggests that enantiomeric biochemical events such as 1-MeTIQ biosynthesis may participate in the pathogenesis of Parkinson's disease.     

Co-expression/co-location of S100 proteins (S100B, S100A1 and S100A2) and protein kinase C (PKC-beta, -eta and -zeta) in a rat model of cerebral basilar artery vasospasm

OBJECT: The cellular events leading to cerebral vasospasm after subarachnoid haemorrhages (SAH) involve a number of members of the protein kinase C (PKC) family. However, whereas calcium is thought to play a number of major roles in the pathophysiology of SAH, a number of PKCs function independently of calcium. We recently emphasized the potential role of the calcium-binding S100 proteins in a 'double haemorrhage' rat model of SAH-induced vasospasm. A number of S100 proteins are known to interfere directly with PKC, or indirectly with PKC substrates. We therefore investigated whether specific S100 proteins and PKCs are co-expressed/co-located in a rat model of SAH-induced vasospasm. METHODS AND RESULTS: SAH-induced vasospasm in rats (by means of a double cisternal injection of autologous blood from a rat femoral artery) distinctly modified the expression levels of calcium-dependent PKC-alpha and PKC-beta and calcium-independent PKC-eta and PKC-zeta in endothelial and smooth-muscle cells. The RNA levels of these four PKC isotypes were determined by quantitative RT-PCR. The present study reveals that, in endothelial cells, the S100B expression/location correlate well with those of PKC-eta, and those of S100A1 with PKC-beta. In smooth-muscle cells S100A2 expression/location correlate with those of PKC-eta, and those of S100B with PKC-zeta. CONCLUSION: The present data argue in favour of a joint action of the S100 protein network and the PKC signalling pathway during cerebral vasospasm.     

MDR1基因及P—gp在脑胶质瘤中定量表达的研究

目的检测脑胶质瘤中多药耐药基因ＭＤＲ１、ｍＲＮＡ和膜糖蛋白Ｐ－ｇｐ相对含量，确定胶质瘤的化疗敏感性。方法免疫组织化学和逆转录聚合酶链反应（ＲＴ－ＰＣＲ）。结果在非化疗组和短期化疗组ＭＤＲ１ｍＲＮＡ含量及Ｐ－ｇｐ表达无显著差别，但长期化疗组明显高于前两组。结论推测ＭＤＲ１基因过度表达可能是胶质瘤继发耐药的主要原因。Ｐ－ｇｐ的表达程度与ＭＤＲ１ｍＲＮＡ含量相关     

Coenzyme Q(1) depletes NAD(P)H and impairs recycling of ascorbate in astrocytes

Ascorbate is an important antioxidant in the brain. Astrocytes are capable of recycling ascorbate by taking up and then reducing its oxidation product dehydroascorbic acid (DHAA) using reducing equivalents derived from NAD(P)H. Astrocytes also contain NAD(P)H-dependent quinone reductases, such as NAD(P)H:quinone oxidoreductase (NQO1), which are capable of reducing coenzyme Q and its analogs. Short-chain coenzyme Q analogs have been proposed as therapeutic agents for neurodegenerative illnesses, but they may cause oxidative stress by non-enzymatic redox cycling or enzyme-dependent depletion of NAD(P)H. Therefore, we tested the hypothesis that the short-chain coenzyme Q analog coenzyme Q(1) (CoQ(1), ubiquinone-5) decreases intracellular NAD(P)H levels in astrocytes and impairs the ability of these cells to replace extracellular DHAA with ascorbate (i.e., ascorbate recycling). We observed that CoQ(1) inhibited the production of intra- and extracellular ascorbate by primary rat astrocytes incubated with DHAA in glucose-free medium. Reduction of CoQ(1) to CoQ(1)H(2) by astrocytes was partially blocked by the NQO1 inhibitor dicumarol but was not affected by DHAA. The inhibition of ascorbate recycling by CoQ(1) was attenuated by dicumarol and was abolished by glucose. CoQ(1) lowered intracellular levels of reactive oxygen species, as measured by oxidation of 2',7'-dichlorofluorescin but also produced marked decreases in the concentrations of NADH and NADPH. We conclude that in astrocytes CoQ(1) recycling depletes NAD(P)H and inhibits ascorbate recycling when glucose metabolism is limited. Because DHAA can cause cell-lethal oxidative stress in neurons and ascorbate produced by astrocytes may be neuroprotective, coenzyme Q analogs may adversely affect brain function through this novel mechanism.     

Poly(ADP-ribose) polymerase-1 activation in a primate model of multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated disabling neurological disorder involving inflammation, demyelination, axonal damage, and neurodegeneration. Poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme linked to DNA repair, has been shown to regulate the cellular inflammatory response through interactions with nuclear factor-kappaB. Extensive PARP-1 activation can, by separate mechanisms, also cause cell death. PARP-1 activation in brain occurs in several settings associated with oxidative stress and DNA damage, and PARP-1 inhibition has been shown to attenuate inflammation and improve neuronal survival in these settings. Here we studied the pattern of PARP-1 activation in a nonhuman primate model of MS, marmoset (Callithrix jacchus) experimental allergic encephalomyelitis (EAE). Characteristic of this model is relapsing and remitting focal demyelination typical of human MS. Immunostaining for poly(ADP-ribose), the enzymatic product of PARP-1, showed PARP-1 activation specifically in plaque areas of EAE brains. Robust immunostaining was found in astrocytes surrounding demyelinated EAE plaques and in scattered nearby microglia, oligodendrocytes, and neurons. The immunostaining also suggested PARP-1 activation in occasional endothelial cells surrounded by microglia or infiltrating peripheral blood cells. Given the importance of PARP-1 in both inflammation and cell death processes, these findings suggest that PARP-1 activation may be a significant factor in the pathogenesis of MS.     

Dysfunctions of automatic (P300a) and controlled (P300b) processing in Parkinson’s disease

Abstract

P300 Event Related Potentials components (P300a and P300b) were investigated using an auditory oddball paradigm (with abutton press response to target stimuli) in 15 Parkinson’s disease patients and 50 normal controls whilst simultaneously measuring electrodermal activity. Cluster analysis showed that the first 10 target stimuli generated the largest skin conductance responses. The first 10 single-trial ERP epochs were therefore analysed as an ERP sub-average for each individual. The P300a component (associated with the automatic ‘Orienting Reflex’) was expected to be most prevalent in this sub-average (compared with subaverages of subsequent blocks of 10 target stimuliJ. Twenty-nine out of50 normal controls (58%) elicited a P300a in the first 10 target sub-averages compared with only 2 out of 15 Parkinson’s disease subjects (J 3%). The conventional P300b component (associated with controlled processing) was found to be significantly delayed for all sub-averages for the Parkinson’s disease group when compared with controls. These preliminary findings suggest a possible dysfunction in both automatic and controlled processing in this disorder. [Neural Res 1998: 20: 5–10]     

Loss of tuberous sclerosis complex 1 (Tsc1) expression results in increased Rheb/S6K pathway signaling important for astrocyte cell size regulation

Individuals with tuberous sclerosis complex (TSC) develop central nervous system abnormalities that may reflect astrocyte dysfunction. In an effort to model astrocyte dysfunction in TSC, we generated mice lacking Tsc1 (hamartin) expression in astrocytes and demonstrated that Tsc1-null astrocytes exhibit abnormalities in contact inhibition growth arrest. In this study, we demonstrate that hamartin-deficient astrocytes are also defective in cell size regulation. We show that the increase in Tsc1-null astrocyte size is associated with increased activation of the S6-kinase pathway. In keeping with recent reports that the hamartin/tuberin complex may regulate Rheb and downstream S6K activation, we demonstrate that expression of either Rheb or S6K in primary astrocytes results in increased S6 pathway activation, and that inhibition of Rheb activity in Tsc1-deficient astrocytes using either pharmacologic or genetic strategies markedly reduces S6 activation. Collectively, these observations suggest that TSC inactivation in astrocytes results in defective cell size regulation associated with dysregulated Rheb/mTOR/S6K pathway activity.     

Thrombotic safety of prothrombin complex concentrate (Beriplex P/N) for dabigatran reversal in a rabbit model

Introduction

In vivo animal data have shown prothrombin complex concentrate (PCC) to be effective in preventing bleeding induced by excessive plasma levels of the direct thrombin inhibitor dabigatran. This animal model study was designed to determine the risk of thrombosis associated with administration of a PCC (Beriplex P/N) to reverse dabigatran-induced bleeding.

Materials and methods

Anesthetized rabbits were treated with initial 0, 75, 200 or 450 μg kg^- 1 dabigatran boluses followed by continuous infusions to maintain elevated plasma dabigatran levels. At 15 min after the start of dabigatran administration, PCC doses of 0, 50 or 300 IU kg^- 1 were administered. Thereafter, coagulation in an arteriovenous (AV) shunt was evaluated and histopathologic examination for thrombotic changes performed. Venous thrombosis was also assessed in a modified Wessler model.

Results

At the suprapharmacologic dose of 300 IU kg^- 1, PCC increased thrombus weight during AV shunting, but this effect could be prevented by dabigatran at all tested doses. AV shunt occlusion after PCC administration was delayed by 75 μg kg^- 1 dabigatran and abolished by progressively higher dabigatran doses. High-dose treatment with 300 IU kg^- 1 PCC resulted in histologically evident low-grade pulmonary thrombi; however, that effect could be blocked by dabigatran in a dose-dependent manner (p = 0.034). In rabbits treated with high-dose PCC, dabigatran inhibited thrombus formation during venous stasis. PCC effectively reversed dabigatran-induced bleeding.

Conclusions

In this animal study, thrombosis after PCC administration could be prevented in the presence of dabigatran. PCC reversed dabigatran-induced excessive bleeding while retaining protective anticoagulatory activity of dabigatran.     

Characteristics of membrane progestin receptor alpha (mPRα) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions

Rapid, progestin actions initiated at the cell surface that are often nongenomic have been described in a variety of reproductive tissues, but until recently the identities of the membrane receptors mediating these nonclassical progestins actions remained unclear. Evidence has been obtained in the last 4–5 years for the involvement of two types of novel membrane proteins unrelated to nuclear steroid receptors, progesterone membrane receptors (mPRs) and progesterone receptor membrane component 1 (PGMRC1), in progestin signaling in several vertebrate reproductive tissues and in the brain. The mPRs, (M_W 40 kDa) initially discovered in fish ovaries, comprise at least three subtypes, α, β and γ and belong to the seven-transmembrane progesterone adiponectin Q receptor (PAQR) family. Both recombinant and wildtype mPRs display high affinity (K_d 5 nM), limited capacity, displaceable and specific progesterone binding. The mPRs are directly coupled to G proteins and typically activate pertussis-sensitive inhibitory G proteins (G_i), to down-regulate adenylyl cyclase activity. Recent studies suggest the alpha subtype (mPRα) has important physiological functions in variety of reproductive tissues. The mPRα is an intermediary in progestin induction of oocyte maturation and stimulation of sperm hypermotility in fish. In mammals, the mPRαs have been implicated in progesterone regulation of uterine function in humans and GnRH secretion in rodents. The single-transmembrane protein PGMRC1 (M_W 26–28 kDa) was first purified from porcine livers and its cDNA was subsequently cloned from porcine smooth muscle cells and a variety of other tissues by different investigators. PGMRC1 and the closely-related PGMRC2 belong to the membrane-associated progesterone receptor (MAPR) family. The PGMRC1 protein displays moderately high binding affinity for progesterone which is 2- to 10-fold greater than that for testosterone and glucocorticoids, and also can bind to other molecules such as heme, cholesterol metabolites and proteins. The signal transduction pathways induced by binding of progesterone to PGMRC1 have not been described to date, although motifs for tyrosine kinase, kinase binding, SH2 and SH3 have been predicted from the amino acid sequence. Evidence has been obtained that PGMRC1 mediates the antiapoptotic affects of progesterone in rat granulosa cells. The PGMRC1 protein may also be an intermediary in the progesterone induction of the acrosome reaction in mammalian sperm. Despite these recent advances, many aspects of progestin signaling through these two families of novel membrane proteins remain unresolved. Biochemical characterization of the receptors has been hampered by rapid degradation of the partially purified proteins. A major technical challenge has been to express sufficient amounts of the recombinant receptors on the plasma membranes in eukaryotic systems to permit investigations of their progestin binding and signal transduction characteristics. Additional basic information on the molecular and cellular mechanisms by which mPRs and PGMRC1 interact with progestins, signal transductions pathways and other proteins will be required to establish a comprehensive model of nontraditional progestin actions mediated through these novel proteins.     

Mild moxibustion at Tianshu (ST 25) decreases expression of prokineticin-1 and prokineticin receptor-1 in colon tissue of rats with chronic visceral hyperalgesia

Prokineticin-1 and prokineticin receptor-1 play important roles in visceral hypersensitivity and in-flammatory pain. Visceral hypersensitivity is closely associated with irritable bowel syndrome. Mild moxibustion can relieve chronic visceral hyperalgesia in rats with irritable bowel syndrome. We hypothesized that prokineticin-1 and prokineticin receptor-1 is the key target in the mechanism. This study established chronic visceral hyperalgesia rat models by colorectal distention. Protein and mRNA expression ...     

Substance P (SP) and neurokinin a (NKA) in developing submandibular glands of the rat

The effect of isoprenaline, carbachol, substance P (SP) and neurokinin A (NKA) on peroxidase and total protein secretion was studied in the developing postnatal submandibular glands of the rat using in vitro methods. Submandibular glands of 1, 5, 12 and 30 day-old-rats were stimulated by 10⁻⁵ M isoprenaline and carbachol, and 10⁻⁶ M SP and NKA. The stimulatory effects of these compounds were compared to the basic release of peroxidase and total amount of protein from submandibular gland fragments in incubation solution with no added transmitter substances. Indirect immunohistochemical methods were used to study these developing glands from SP- and NKA-immunoreactive (IR) nerve fibers. The distributions of SP-IR and NKA-IR nerve fibers closely resembled each other, being most abundantly spread around the developing acini and ducts. The number of these fibers was high on the 1st, 5th and 12th days, but was decreased on the 30th day. On peroxidase release, isoprenaline was the most effective, causing a maximal response of 47 times the basic release on the first postnatal day, after which it gradually decreased. The effects of carbachol, SP and NKA on peroxidase release were clearly weaker and, unlike isoprenaline, their strongest response was on the 5th postnatal day (carbachol, 4.3; SP 5.2; NKA, 4.5). The total protein secretion effect patterns of the studied substances resembled each other more, showing their strongest response on the 5th day (isoprenaline, 5.0; carbachol, 4.5; SP, 4.2; NKA, 3.4) and decreasing thereafter.In general, both the stimulatory pattern of SP and NKA, and the appearance and distribution of SP-IR and NKA-IR nerve fibers during the early postnatal period of development closely resemble those of parasympathetic nerves and their transmitters. Furthermore, it is concluded that the abundancy of SP-IR and NKA-IR nerves in the developing submandibular gland is accompanied by the increased sensitivity of the secretory elements to these tachykinins, indicating that they play a role in the maturation of the glandular secretory functions.     

Alpha-smooth muscle actin (alpha-SMA) and nestin expression in reactive astrocytes in multiple sclerosis lesions: potential regulatory role of transforming growth factor-beta 1 (TGF-beta1)

Aims: Rapid and extensive activation of astrocytes occurs subsequent to many forms of central nervous system (CNS) injury. Recent studies have revealed that the expression profile of reactive astrocytes comprises antigens present during astrocyte development. Elevated levels of the injury‐related cytokine transforming growth factor‐beta 1 (TGF‐β1) secreted by microglial cells and invading macrophages have been correlated with the reactive astrocyte phenotype and glial scar formation. Methods: In the present study, the expression profile of alpha‐smooth muscle actin (α‐SMA) and nestin, two cytoskeletal proteins expressed during astrocyte development, was studied in multiple sclerosis (MS) lesions. In addition, α‐SMA and nestin organization and expression were analysed in rat primary astrocyte cultures in response to TGF‐β1. Results: In active lesions and in the hypercellular margin of chronic active MS lesions, immunostaining for α‐SMA revealed a subpopulation of reactive astrocytes, whereas the majority of reactive astrocytes expressed nestin. α‐SMA and nestin expressing reactive astrocytes were in close relationship with TGF‐β1 expressing macrophages or microglia. In addition, TGF‐β1 expression within α‐SMA or nestin expressing astrocytes was also detected. Our in vitro experiments showed that TGF‐β1 regulated the organization and expression of α‐SMA and nestin in astrocytes. Conclusions: Reactive astrocytes in active MS lesions re‐express α‐SMA and nestin. We suggest that the in vivo re‐expression might be under regulation of TGF‐β1. These results further clarify the regulation of astrocyte activity after CNS injury, which is important for the astroglial adaptation to pathological situations.