Identification of early disease progression in an ALS rat model

Transgenic rat models of amyotrophic lateral sclerosis (ALS) have recently been developed. Most assays of ALS-symptoms in these models monitor disease onset accurately, but do not identify individuals that will develop these symptoms before the motor deficits become apparent. Peak bodyweight has recently been shown to indicate affected individuals before motor deficits become apparent. However, it must be determined retrospectively due to weight fluctuation. Here, we report that exploratory activities detected by a photobeam activity system (PAS) and wire mesh ascending test can be used to detect slight motor deficits in the early phase of ALS. Thus, these tests may be used in addition to peak bodyweight to monitor early disease progression and to assay efficacy of new therapeutic interventions.     

Inhibition of calpains, by treatment with leupeptin, improves motoneuron survival and muscle function in models of motoneuron degeneration

The effect of treatment with leupeptin, a calpain inhibitor, on motoneuron survival and muscle function was examined in in vitro and in vivo models of motoneuron degeneration. Exposure of primary rat motoneurons to alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) is an established in vitro model of excitotoxic motoneuron death. Here we show that leupeptin treatment improved motoneuron survival following exposure to AMPA (50 microM). Application of leupeptin (100 microM) to AMPA treated cultures rescued many motoneurons so that 74% (+/-3.4 S.E.M., n=5) survived compared with only 49% (+/-2.4 S.E.M., n=5) in untreated cultures. The effect of treatment with leupeptin on motoneuron survival and muscle function was also examined in vivo. In 3 day-old rats, the sciatic nerve was crushed and at the time of injury, a silicon implant containing leupeptin was inserted onto the lumbar spinal cord. The effect on long-term motoneuron survival and muscle function was assessed 12 weeks after injury. The results showed that there was long-term improvement in motoneuron survival in the leupeptin treated group. Thus, in untreated animals 12 weeks after nerve crush only 30% (+/-2.8. S.E.M., n=3) of sciatic motoneurons survived compared with 43% (+/-1.5 S.E.M., n=3) in the leupeptin-treated group. This improvement in motoneuron survival was reflected in a significant improvement in muscle function in the leupeptin-treated group. For example in the soleus muscle of treated rats 20.8 (+/-1.40 S.E.M., n=5) motor units survived compared with only 14.6 (+/-1.21 S.E.M., n=5) in untreated animals. Thus, treatment with leupeptin, a calpain inhibitor, rescues motoneurons from cell death and improves muscle function following nerve injury.     

Ionic current model of a hypoglossal motoneuron

We have developed a single-compartment, electrophysiological, hypoglossal motoneuron (HM) model based primarily on experimental data from neonatal rat HMs. The model is able to reproduce the fine features of the HM action potential: the fast afterhyperpolarization, the afterdepolarization, and the medium-duration afterhyperpolarization (mAHP). The model also reproduces the repetitive firing properties seen in neonatal HMs and replicates the neuron's response to pharmacological experiments. The model was used to study the role of specific ionic currents in HM firing and how variations in the densities of these currents may account for age-dependent changes in excitability seen in HMs. By varying the density of a fast inactivating calcium current, the model alternates between accelerating and adapting firing patterns. Modeling the age-dependent increase in H current density accounts for the decrease in mAHP duration observed experimentally, but does not fully account for the decrease in input resistance. An increase in the density of the voltage-dependent potassium currents and the H current is required to account for the decrease in input resistance. These changes also account for the age-dependent decrease in action potential duration.     

Early signs of motoneuron vulnerability in a disease model system: Characterization of transverse slice cultures of spinal cord isolated from embryonic ALS mice

Mutations in the SOD1 gene are associated with familial amyotrophic lateral sclerosis. The mechanisms by which these mutations lead to cell loss within the spinal cord ventral horns are unknown. In the present report we used the G93A transgenic mouse model of amyotrophic lateral sclerosis to develop and characterize an in vitro tool for the investigation of subtle alterations of spinal tissue prior to frank neuronal degeneration. To this aim, we developed organotypic slice cultures from wild type and G93A embryonic spinal cords. We combined immunocytochemistry and electron microscopy techniques to compare wild type and G93A spinal cord tissues after 14 days of growth under standard in vitro conditions. By SMI32 and choline acetyl transferase immunostaining, the distribution and morphology of motoneurons were compared in the two culture groups. Wild type and mutant cultures displayed no differences in the analyzed parameters as well as in the number of motoneurons. Similar results were observed when glial fibrillary acidic protein and myelin basic protein-positive cells were examined. Cell types within the G93A slice underwent maturation and slices could be maintained in culture for at least 3 weeks when prepared from embryos. Electron microscopy investigation confirmed the absence of early signs of mitochondria vacuolization or protein aggregate formation in G93A ventral horns. However, a significantly different ratio between inhibitory and excitatory synapses was present in G93A cultures, when compared with wild type ones, suggesting the expression of subtle synaptic dysfunction in G93A cultured tissue. When compared with controls, G93A motoneurons exhibited increased vulnerability to AMPA glutamate receptor-mediated excitotoxic stress prior to clear disease appearance. This in vitro disease model may thus represent a valuable tool to test early mechanisms contributing to motoneuron degeneration and potential therapeutic molecular interventions.     

X射线引导建立大鼠椎间盘退变模型

背景:目前国内外对于椎间盘退变的造模方法较多,但存在定位不精准问题.目的:对比X射线引导下与非X射线引导下建立大鼠椎间盘退变模型的效果.方法:取9只成年SD大鼠,按照随机数字表法分3组,每组3只,空白组不进行任何操作;对照组手动定位尾椎Co6/7椎间盘,用注射器针头进行垂直穿刺,并均匀缓慢注入无水乙醇,建立椎间盘退变模...     

Feeding induced by intracerebroventricular 2-deoxy-D-glucose in the rat.

Feeding was induced with lateral intracerebroventricular (ICV) injections of 2-deoxy-D-glucose (2-dg), a glucose analogue causing glucoprivation. Feeding increased rapidly by an average of 2.6 g following ICV 2-DG (2.9 or 5.8 mg per rat), but did not increase after ICV D-glucose or sucrose. At the same doses, 2-DG did not increase feeding when given peripherally. Core temperature dropped in a dose-dependent manner with doses of 2-DG sufficient to induce feeding after peripheral administration, but did not occur with ICV injections. The 2-DG (0.006--1.219 M) did not stimulate feeding when infused bilaterally into the lateral hypothalamus, the preoptic area, or the anterior lateral hypothalamus. Nor did it produce feeding when injected into the ventromedial hypothalamus at the same sites and in the same rats in which procaine HC1 caused increased feeding. The brain, therefore, is directly sensitive to glucoprivation in the control of feeding, and glucoprivation alone is sufficient to mobilize feeding behavior. The specific site of sensitivity to glucoprivation and the mode of action of the glucoprivic system in the brain are unknown.     

Lipolytic responses induced by intracerebroventricular administration of histamine in the rat

Histamine (10–50 g) administered intraventricularly in conscious rats induced an increase in serum-free fatty acids. The maximum, significant increase appeared 30–60 min after administration. Histamine H₁-receptor antagonists, mepyramine and chloropyramine, when injected 2 h prior to histamine, abolished considerably hyperlipaemic responses to histamine. H₂-Receptor antagonists, metiamide and cimetidine, given i.c.v. only moderately diminished the histamine-induced hyperlipaemia. Histamine injected i.c.v. also increased serum corticosterone levels considerably. This elevation was prevented significantly by the H₁-receptor antagonist, mepyramine, but not by the H₂-receptor blocker, cimetidine. It seems likely that histamine given i.c.v. induces lipolysis through the release of ACTH, one of the known lipid-mobilizing hormones. The central lipid-mobilizing mechanism after histamine depends more on activation of H₁- than H₂-receptors.     

Retinal blood vessels are damaged in a rat model of NMDA-induced retinal degeneration

Retinal ischemia–reperfusion causes capillary degeneration but the mechanisms of damage are not well understood. The NMDA receptor plays an important role in neuronal damage after ischemia–reperfusion. Therefore, we determined whether retinal blood vessels are damaged structurally and functionally in a rat model of retinal degeneration induced by NMDA. At 7 days after a single intravitreal injection of NMDA (200 nmol) into the eye, loss of retinal ganglion cells and thinning of the inner plexiform layer were observed. Endothelial cells disappeared in some regressing vessels and empty basement membrane sleeves were left as remnants of the vessels. The number of basement membrane sleeves was increased in the NMDA-treated retina and non-perfused vessels were found in the injured retina. These results indicate that retinal blood vessels are damaged in the NMDA-induced retinal degeneration model. Neuronal cells may play a role in maintaining normal structure and function of the vasculature in the retina.     

VEGF和bFGF在大鼠压疮模型中的表达及其意义

目的:研究血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)与压疮的关系,从分子水平为有效防治压疮提供理论依据。方法:将SD大鼠随机分成对照组和实验组,采用磁片循环压迫法建立大鼠Ⅲ度压疮模型,HE染色观察大鼠皮肤组织病理学变化,免疫组织化学方法检测标本中VEGF和bFGF蛋白表达情况,分析二者变化的一致性。结果:VEGF和bFGF蛋白在大鼠Ⅲ度压疮组织中的表达低于压疮周边组织及正常大鼠皮肤(P＜0.01),且VEGF和bFGF的变化呈一致性(κ=0.58)。结论:VEGF和bFGF在大鼠Ⅲ度压疮组织表达降低,提示其可能是Ⅲ度压疮皮肤难以愈合的潜在关键因素。     

Intracranial MEMS based temozolomide delivery in a 9L rat gliosarcoma model

Primary malignant brain tumors (BT) are the most common and aggressive malignant brain tumor. Treatment of BTs is a daunting task with median survival just at 21 months. Methods of localized delivery have achieved success in treating BT by circumventing the blood brain barrier and achieving high concentrations of therapeutic within the tumor. The capabilities of localized delivery can be enhanced by utilizing mirco-electro-mechanical systems (MEMS) technology to deliver drugs with precise temporal control over release kinetics. An intracranial MEMS based device was developed to deliver the clinically utilized chemotherapeutic temozolomide (TMZ) in a rodent glioma model. The device is a liquid crystalline polymer reservoir, capped by a MEMS microchip. The microchip contains three nitride membranes that can be independently ruptured at any point during or after implantation. The kinetics of TMZ release were validated and quantified in?vitro. The safety of implanting the device intracranially was confirmed with preliminary in?vivo studies. The impact of TMZ release kinetics was investigated by conducting in?vivo studies that compared the effects of drug release rates and timing on animal survival. TMZ delivered from the device was effective at prolonging animal survival in a 9L rodent glioma model. Immunohistological analysis confirmed that TMZ was released in a viable, cytotoxic form. The results from the in?vivo efficacy studies indicate that early, rapid delivery of TMZ from the device results in the most prolonged animal survival. The ability to actively control the rate and timing of drug(s) release holds tremendous potential for the treatment of BTs and related diseases.     

Differential brain,but not serum VEGF levels in a genetic rat model of depression

Compared to the classical monoamine hypotheses focus on neuroplasticity is a major new approach in studies of depression and antidepressants. Recent studies have demonstrated that vascular endothelial growth factor (VEGF) is regulated by antidepressant treatment in rodents. However, in depressive patients no significant changes were found in the serum VEGF levels compared to control subjects. To our knowledge, brain and serum VEGF levels have never been reported in parallel for any psychiatric disease model. That prompted us to examine the levels of VEGF in serum, hippocampus, frontal cortex, corpus striatum, and hypothalamus in male Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL), a genetic rat model of depression. The VEGF levels were identical in the FSL and the FRL rats in serum, corpus striatum, and hypothalamus. In hippocampus and frontal cortex, the VEGF levels were significantly decreased in the FSL rats compared to the FRL rats. The results may add to the hypothesis that altered expression of growth factors/neurotrophic factors are related to the pathophysiology of depression.     

A novel rat tail disc degeneration model induced by static bending and compression

BackgroundA new rat tail intervertebral disc degeneration model was established to observe the morphologic and biologic changes of static bending and compression applied to the discs.MethodsIn total, 20 Sprague‐Dawley rats with similar weight were randomly divided into 4 groups. Group 1 served as a control group for a baseline assessment of normal discs. Group 2 underwent a sham surgery, using an external device to bend the vertebrae of coccygeal 8‐10. Groups 3 and 4 were the loaded groups, and external devices were instrumented to bend the spine with a compression level of 1.8 N and 4.5 N, respectively. Magnetic resonance imaging (MRI), histological, and quantitative real‐time PCR (qRT‐PCR) analysis were performed on all animals on day 14 of the experiment.ResultsMagnetic resonance imaging and histological results showed that the changes of intervertebral disc degeneration increased with the size of compression load. Some architecture disorganizations in nucleus pulposus and annulus fibrosus were found on both of the convex and concave side in the groups of 1.8 N and 4.5 N. An upregulation of MM‐3, MM‐13, and collagen 1‐α1 mRNA expression and a downregulation of collagen 2‐α1 and aggrecan mRNA expression were observed in the sham and loading groups. Significant changes were found between the loading groups, whereas the sham group showed similar results to the control group.ConclusionsStatic bending and compression could induce progressive disc degeneration, which could be used for biologic study on disc degeneration promoted by static complex loading.     

Hepatocyte growth factor (HGF) attenuates gliosis and motoneuronal degeneration in the brainstem motor nuclei of a transgenic mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of brainstem and spinal motoneurons. Although prevention of motoneuronal degeneration has been postulated as the primary target for a cure, accumulating evidence suggests that microglial accumulation contributes to disease progression. This study was designed to assess the ability of HGF to modulate microglial accumulation and motoneuronal degeneration in brainstem motor nuclei, using double transgenic mice overexpressing mutated SOD1^G93A and HGF (G93A/HGF). Histological and immunohistochemical analyses of the tissues of G93A/HGF mice revealed a marked decrease in the number of microglia and reactive astrocytes and an attenuation of the loss of motoneurons in facial and hypoglossal nuclei compared with G93A mice. HGF overexpression attenuated monocyte chemoattractant protein-1 (MCP-1) induction, predominantly in astrocytes; suppressed activation of caspase-1, -3 and -9; and, increased X chromosome-linked inhibition of apoptosis protein (XIAP) in the motoneurons of G93A mice. The implication is that HGF reduces microglial accumulation by suppressing MCP-1 induction and prevents motoneuronal death through inhibition of pro-apoptotic protein activation. These findings suggest that, in addition to direct neurotrophic activity on motoneurons, HGF-suppression of gliosis may retard disease progression, making HGF a potential therapeutic agent for the treatment of ALS patients.     

Hepatocyte growth factor (HGF) attenuates gliosis and motoneuronal degeneration in the brainstem motor nuclei of a transgenic mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of brainstem and spinal motoneurons. Although prevention of motoneuronal degeneration has been postulated as the primary target for a cure, accumulating evidence suggests that microglial accumulation contributes to disease progression. This study was designed to assess the ability of HGF to modulate microglial accumulation and motoneuronal degeneration in brainstem motor nuclei, using double transgenic mice overexpressing mutated SOD1(G93A) and HGF (G93A/HGF). Histological and immunohistochemical analyses of the tissues of G93A/HGF mice revealed a marked decrease in the number of microglia and reactive astrocytes and an attenuation of the loss of motoneurons in facial and hypoglossal nuclei compared with G93A mice. HGF overexpression attenuated monocyte chemoattractant protein-1 (MCP-1) induction, predominantly in astrocytes; suppressed activation of caspase-1, -3 and -9; and, increased X chromosome-linked inhibition of apoptosis protein (XIAP) in the motoneurons of G93A mice. The implication is that HGF reduces microglial accumulation by suppressing MCP-1 induction and prevents motoneuronal death through inhibition of pro-apoptotic protein activation. These findings suggest that, in addition to direct neurotrophic activity on motoneurons, HGF-suppression of gliosis may retard disease progression, making HGF a potential therapeutic agent for the treatment of ALS patients.     

Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease

Several lines of evidence point to a significant role of neuroinflammation in Parkinson's disease (PD) and other neurodegenerative disorders. In the present study we examined the protective effect of celecoxib, a selective inhibitor of the inducible form of cyclooxygenase (COX-2), on dopamine (DA) cell loss in a rat model of PD. We used the intrastriatal administration of 6-hydroxydopamine (6-OHDA) that induces a retrograde neuronal damage and death, which progresses over weeks. Animals were randomized to receive celecoxib (20 mg/kg/day) or vehicle starting 1 hour before the intrastriatal administration of 6-OHDA. Evaluation was performed in vivo using micro PET and selective radiotracers for DA terminals and microglia. Post mortem analysis included stereological quantification of tyrosine hydroxylase, astrocytes and microglia. 12 days after the 6-OHDA lesion there were no differences in DA cell or fiber loss between groups, although the microglial cell density and activation was markedly reduced in animals receiving celecoxib (p < 0.01). COX-2 inhibition did not reduce the typical astroglial response in the striatum at any stage. Between 12 and 21 days, there was a significant progression of DA cell loss in the vehicle group (from 40 to 65%) that was prevented by celecoxib. Therefore, inhibition of COX-2 by celecoxib appears to be able, either directly or through inhibition of microglia activation to prevent or slow down DA cell degeneration.     

Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease

Molecular hydrogen serves as an antioxidant that reduces hydroxyl radicals, but not the other reactive oxygen and nitrogen species. In the past year, molecular hydrogen has been reported to prevent or ameliorate eight diseases in rodents and one in human associated with oxidative stress. In Parkinson's disease, mitochondrial dysfunction and the associated oxidative stress are major causes of dopaminergic cell loss in the substantia nigra. We examined effects of ∼50%-saturated molecular hydrogen in drinking water before or after the stereotactic surgery on 6-hydroxydopamine-induced nigrostrital degeneration in a rat model of Parkinson's disease. Methamphetamine-induced behavioral analysis showed that molecular hydrogen prevented both the development and progression of the nigrostrital degeneration. Tyrosine hydroxylase staining of the substantia nigra and striatum also demonstrated that pre- and post-treatment with hydrogen prevented the dopaminergic cell loss. Our studies suggest that hydrogen water is likely able to retard the development and progression of Parkinson's disease.     

犬椎间盘经皮针刺退变模型的建立

背景：普通针头穿刺纤维环是目前较为公认的理想椎间退变模型制作方法,但存在对动物损伤大,操作不简捷等缺点。 目的：尝试在C臂机透视下经皮穿刺比格犬腰椎椎间隙建立腰椎间盘退变动物模型。 方法：选用8只健康成年犬,行腰椎MRI后在C臂机下经皮穿刺椎间隙,损伤L5/6椎间盘。术后3,6个月行腰椎MRI后,取标本行免疫组织化学观察椎间盘退变情况。 结果与结论：经皮椎间盘穿刺后3,6个月,L5/6椎间盘均出现了明显退变,MRI显示T2值均出现明显降低,并且椎间盘大多出现不同程度椎间盘突出征象;L5/6穿刺节段髓核Ⅱ型胶原阳性细胞含量较L4/5髓核均明显减少(P < 0.05)。说明经皮穿刺损伤椎间盘建立犬椎间盘退变模型是一种简单有效、重复性好的建模方法。     

Synthetic biodegradable hydrogel delivery of demineralized bone matrix for bone augmentation in a rat model

There exists a strong clinical need for a more capable and robust method to achieve bone augmentation, and a system with fine-tuned delivery of demineralized bone matrix (DBM) has the potential to meet that need. As such, the objective of the present study was to investigate a synthetic biodegradable hydrogel for the delivery of DBM for bone augmentation in a rat model. Oligo(poly(ethylene glycol) fumarate) (OPF) constructs were designed and fabricated by varying the content of rat-derived DBM particles (either 1:3, 1:1 or 3:1 DBM:OPF weight ratio on a dry basis) and using two DBM particle size ranges (50–150 or 150–250 μm). The physical properties of the constructs and the bioactivity of the DBM were evaluated. Selected formulations (1:1 and 3:1 with 50–150 μm DBM) were evaluated in vivo compared to an empty control to investigate the effect of DBM dose and construct properties on bone augmentation. Overall, 3:1 constructs with higher DBM content achieved the greatest volume of bone augmentation, exceeding 1:1 constructs and empty implants by 3- and 5-fold, respectively. As such, we have established that a synthetic, biodegradable hydrogel can function as a carrier for DBM, and that the volume of bone augmentation achieved by the constructs correlates directly to the DBM dose.     

Selective vulnerability and pruning of phasic motoneuron axons in motoneuron disease alleviated by CNTF

Neurodegenerative diseases can have long preclinical phases and insidious progression patterns, but the mechanisms of disease progression are poorly understood. Because quantitative accounts of neuronal circuitry affected by disease have been lacking, it has remained unclear whether disease progression reflects processes of stochastic loss or temporally defined selective vulnerabilities of distinct synapses or axons. Here we derive a quantitative topographic map of muscle innervation in the hindlimb. We show that in two mouse models of motoneuron disease (G93A SOD1 and G85R SOD1), axons of fast-fatiguable motoneurons are affected synchronously, long before symptoms appear. Fast-fatigue-resistant motoneuron axons are affected at symptom-onset, whereas axons of slow motoneurons are resistant. Axonal vulnerability leads to synaptic vesicle stalling and accumulation of BC12a1-a, an anti-apoptotic protein. It is alleviated by ciliary neurotrophic factor and triggers proteasome-dependent pruning of peripheral axon branches. Thus, motoneuron disease involves predictable, selective vulnerability patterns by physiological subtypes of axons, episodes of abrupt pruning in the target region and compensation by resistant axons.