AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure.

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.

Necroptotic-Apoptotic Regulation in an Endothelin-1 Model of Cerebral Ischemia.

The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia-reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death.

Role of central endothelin-1 in hyperalgesia, anhedonia, and hypolocomotion induced by endotoxin in male rats.

Sickness syndrome is an adaptive response that can be distinguished by specific signs and symptoms, such as fever and generalized hyperalgesia. Endothelin-1 (ET-1) is produced by inflammatory stimuli, including lipopolysaccharide, and involved in the pathogenesis of inflammation and pain by acting through ETA and ETB receptors. ET-1 also induces fever by acting on the central nervous system. The present study investigated the role of ET-1 in sickness syndrome responses, including hyperalgesia, anhedonia, and hypolocomotion. Intracerebroventricular ET-1 administration induced mechanical and thermal hyperalgesia in rats, which was ameliorated by the ETA receptor antagonist BQ123 and exacerbated by the ETB receptor antagonist BQ788. A cyclooxygenase blocker did not alter hyperalgesia that was induced by ET-1. Lipopolysaccharide administration induced hyperalgesia, and both BQ123 and BQ788 abolished this mechanical hyperalgesia, but the thermal response was only partially blocked. The blockade of ETA receptors in the hypothalamus also abolished lipopolysaccharide-induced mechanical hyperalgesia, and the ETB receptor antagonist did not influence this response. Lipopolysaccharide also induced anhedonia, reflected by lower sucrose preference, and reduced locomotor activity. Both antagonists restored locomotor activity, but only BQ788 reversed the reduction of sucrose preference. These results indicate that ET-1 and both ETA and ETB receptors are involved in various responses that are related to sickness syndrome, including hyperalgesia, anhedonia, and hypolocomotion, that is induced by LPS. Hypothalamic ETA but not ETB receptors are involved in mechanical hyperalgesia that is observed during lipopolysaccharide-induced sickness syndrome.

Focal Ischemic Injury to the Early Neonatal Rat Brain Models Cognitive and Motor Deficits with Associated Histopathological Outcomes Relevant to Human Neonatal Brain Injury.

Neonatal arterial ischemic stroke is one of the more severe birth complications. The injury can result in extensive neurological damage and is robustly associated with later diagnoses of cerebral palsy (CP). An important part of efforts to develop new therapies include the on-going refinement and understanding of animal models that capture relevant clinical features of neonatal brain injury leading to CP. The potent vasoconstrictor peptide, Endothelin-1 (ET-1), has previously been utilised in animal models to reduce local blood flow to levels that mimic ischemic stroke. Our previous work in this area has shown that it is an effective and technically simple approach for modelling ischemic injury at very early neonatal ages, resulting in stable deficits in motor function. Here, we aimed to extend this model to also examine the impact on cognitive function. We show that focal delivery of ET-1 to the cortex of Sprague Dawley rats on postnatal day 0 (P0) resulted in impaired learning in a touchscreen-based test of visual discrimination and correlated with important clinical features of CP including damage to large white matter structures.

microRNA-451a prevents activation of matrix metalloproteinases 2 and 9 in human cardiomyocytes during pathological stress stimulation.

Matrix metalloproteinases (MMP) are important for cardiac remodeling. Recently, microRNA (miR)-451a has been found to inhibit the expression of both MMP-2 and MMP-9 in human malignancies, but its role in cardiomyocytes has not been explored. We hypothesized that miR-451a modulates MMP-2 and MMP-9 levels in human cardiomyocytes. The role of miR-451a on regulation of MMP-2 and MMP-9 was evaluated in two separate pathological models using Cor.4U human inducible pluripotent stem cell-derived cardiomyocytes (hiPS-CMs): 1) endothelin-1 (ET-1), and 2) 48-h hypoxia (1% O2). Both models were transfected with synthetic miR-451a mimics or scramble control. Expression of both mRNA and miR was determined by quantitative real-time polymerase chain reaction and protein activity by (MMP-2/9) activity assay. Bioinformatic analyses were performed using Targetscan 7.1 and STRING 10.5. hiPS-CMs stimulated by hypoxia increased both MMP-2 and MMP-9 expression levels compared with normoxia (P < 0.05), whereas ET-1 stimulation only increased the MMP-9 level compared with vehicle controls (P < 0.05). miR-451a mimics prevented the increase of MMP-2 and MMP-9 expression in both models. Protein activity of MMP-2 and MMP-9 was confirmed to be lower following treatment with miR-451a mimic compared with scramble-controls. Six of 28 predicted gene transcripts of miR-451a were linked to MMP-2 and MMP-9; Macrophage migration inhibitory factor (MIF) was the only predicted target of miR-451a that was increased by ET-1 and hypoxia and reduced following miR-451a mimic transfection. miR-451a prevent the increase of MMP-2 and MMP-9 in human cardiomyocytes during pathological stress. The modulation by miR-451a on MMP-2 and MMP-9 is caused by MIF.

Mouse connective tissue mast cell proteases tryptase and carboxypeptidase A3 play protective roles in itch induced by endothelin-1.

BACKGROUND: Itch is an unpleasant sensation that can be debilitating, especially if it is chronic and of non-histaminergic origin, as treatment options are limited. Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor that also has the ability to induce a burning, non-histaminergic pruritus when exogenously administered, by activating the endothelin A receptor (ETAR) on primary afferents. ET-1 is released endogenously by several cell-types found in the skin, including macrophages and keratinocytes. Mast cells express ETARs and can thereby be degranulated by ET-1, and mast cell proteases chymase and carboxypeptidase A3 (CPA3) are known to either generate or degrade ET-1, respectively, suggesting a role for mast cell proteases in the regulation of ET-1-induced itch. The mouse mast cell proteases (mMCPs) mMCP4 (chymase), mMCP6 (tryptase), and CPA3 are found in connective tissue type mast cells and are the closest functional homologs to human mast cell proteases, but little is known about their role in endothelin-induced itch. METHODS: In this study, we evaluated the effects of mast cell protease deficiency on scratching behavior induced by ET-1. To investigate this, mMCP knock-out and transgenic mice were injected intradermally with ET-1 and their scratching behavior was recorded and analyzed. RESULTS: CPA3-deficient mice and mice lacking all three proteases demonstrated highly elevated levels of scratching behavior compared with wild-type controls. A modest increase in the number of scratching bouts was also seen in mMCP6-deficient mice, while mMCP4-deficiency did not have any effect. CONCLUSION: Altogether, these findings identify a prominent role for the mast cell proteases, in particular CPA3, in the protection against itch induced by ET-1.

Endothelin-1 contributes to the development of virus-induced demyelinating disease.

BACKGROUND: Experimental autoimmune encephalitis (EAE) and virally induced demyelinating disease are two major experimental model systems used to study human multiple sclerosis. Although endothelin-1 level elevation was previously observed in the CNS of mice with EAE and viral demyelinating disease, the potential role of endothelin-1 in the development of these demyelinating diseases is unknown. METHODS AND RESULTS: In this study, the involvement of endothelin-1 in the development and progression of demyelinating diseases was investigated using these two experimental models. Administration of endothelin-1 significantly promoted the progression of both experimental diseases accompanied with elevated inflammatory T cell responses. In contrast, administration of specific endothelin-1 inhibitors (BQ610 and BQ788) significantly inhibited progression of these diseases accompanied with reduced T cell responses to the respective antigens. CONCLUSIONS: These results strongly suggest that the level of endothelin-1 plays an important role in the pathogenesis of immune-mediated CNS demyelinating diseases by promoting immune responses.

Magnesium acetyltaurate protects against endothelin-1 induced RGC loss by reducing neuroinflammation in Sprague dawley rats.

Endothelin-1 (ET-1), a potent vasoconstrictor, plays a significant role in the pathophysiology of ocular conditions like glaucoma. Glaucoma is characterized by apoptotic loss of retinal ganglion cells (RGCs) and loss of visual fields and is a leading cause of irreversible blindness. In glaucomatous eyes, retinal ischemia causes release of pro-inflammatory mediators such as interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α and promotes activation of transcription factors such as nuclear factor kappa B (NFKB) and c-Jun. Magnesium acetyltaurate (MgAT) has previously been shown to protect against ET-1 induced retinal and optic nerve damage. Current study investigated the mechanisms underlying these effects of MgAT, which so far remain unknown. Sprague dawley rats were intravitreally injected with ET-1 with or without pretreatment with MgAT. Seven days post-injection, retinal expression of IL-1ß, IL-6, TNF-α, NFKB and c-Jun protein and genes was determined using multiplex assay, Western blot and PCR. Animals were subjected to retrograde labeling of RGCs to determine the extent of RGC survival. RGC survival was also examined using Brn3A staining. Furthermore, visual functions of rats were determined using Morris water maze. It was observed that pre-treatment with MgAT protects against ET-1 induced increase in the retinal expression of IL-1ß, IL-6 and TNF-α proteins and genes. It also protected against ET-1 induced activation of NFKB and c-Jun. These effects of MgAT were associated with greater RGC survival and preservation of visual functions in rats. In conclusion, MgAT prevents ET-1 induced RGC loss and loss of visual functions by suppressing neuroinflammatory reaction in rat retinas.

Vascular actions of peripheral CGRP in migraine-like photophobia in mice.

BACKGROUND: Calcitonin gene-related peptide is recognized as a key player in migraine, yet the mechanisms and sites of calcitonin gene-related peptide action remain unknown. The efficacy of calcitonin gene-related peptide-blocking antibodies as preventative migraine drugs supports a peripheral site of action, such as the trigeminovasculature. Given the apparent disconnect between the importance of vasodilatory peptides in migraine and the prevailing opinion that vasodilation is an epiphenomenon, the goal of this study was to test whether vasodilation plays a role in calcitonin gene-related peptide-induced light aversive behavior in mice. METHODS: Systemic mean arterial pressure and light aversive behavior were measured after intraperitoneal administration of calcitonin gene-related peptide and vasoactive intestinal peptide in wild-type CD1 mice. The functional significance of vasodilation was tested by co-administration of a vasoconstrictor (phenylephrine, endothelin-1, or caffeine) with calcitonin gene-related peptide to normalize blood pressure during the light aversion assay. RESULTS: Both calcitonin gene-related peptide and vasoactive intestinal peptide induced light aversion that was associated with their effect on mean arterial pressure. Notably, vasoactive intestinal peptide caused relatively transient vasodilation and light aversion. Calcitonin gene-related peptide-induced light aversion was still observed even with normalized blood pressure. However, two of the agents, endothelin-1 and caffeine, did reduce the magnitude of light aversion. CONCLUSION: We propose that perivascular calcitonin gene-related peptide causes light-aversive behavior in mice by both vasomotor and non-vasomotor mechanisms.

Activation of Cannabinoid Receptors Attenuates Endothelin-1-Induced Mitochondrial Dysfunction in Rat Ventricular Myocytes.

Evidence suggests that the activation of the endocannabinoid system offers cardioprotection. Aberrant energy production by impaired mitochondria purportedly contributes to various aspects of cardiovascular disease. We investigated whether cannabinoid (CB) receptor activation would attenuate mitochondrial dysfunction induced by endothelin-1 (ET1). Acute exposure to ET1 (4 hours) in the presence of palmitate as primary energy substrate induced mitochondrial membrane depolarization and decreased mitochondrial bioenergetics and expression of genes related to fatty acid oxidation (ie, peroxisome proliferator-activated receptor-gamma coactivator-1α, a driver of mitochondrial biogenesis, and carnitine palmitoyltransferase-1ß, facilitator of fatty acid uptake). A CB1/CB2 dual agonist with limited brain penetration, CB-13, corrected these parameters. AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, mediated the ability of CB-13 to rescue mitochondrial function. In fact, the ability of CB-13 to rescue fatty acid oxidation-related bioenergetics, as well as expression of proliferator-activated receptor-gamma coactivator-1α and carnitine palmitoyltransferase-1ß, was abolished by pharmacological inhibition of AMPK using compound C and shRNA knockdown of AMPKα1/α2, respectively. Interventions that target CB/AMPK signaling might represent a novel therapeutic approach to address the multifactorial problem of cardiovascular disease.

Phycocyanobilin reduces brain injury after endothelin-1- induced focal cerebral ischaemia.

Pharmacological therapies for interrupting biochemical events of the ischaemic cascade and protecting against stroke in humans are as yet unavailable. Up to now, the neuroprotective activity in cerebral ischaemia of phycocyanobilin (PCB), a tetrapyrrolic natural antioxidant, has not been fully examined. Here, we evaluated if PCB protects PC12 neuronal cells against oxygen and glucose deprivation plus reperfusion, and its protective effects in a rat model of endothelin-1-induced focal brain ischaemia. PCB was purified from the cyanobacteria Spirulina platensis and characterized by spectrophotometric, liquid and gas chromatography and mass spectrometry techniques. In Wistar rats, PCB at 50, 100 and 200 µg/kg or phosphate-buffered saline (vehicle) was administered intraperitoneally at equal subdoses in a therapeutic schedule (30 minutes, 1, 3 and 6 hours after the surgery). Brain expression of myelin basic protein (MBP) and the enzyme CNPase was determined by immunoelectron microscopy. PCB was obtained with high purity (>95%) and the absence of solvent contaminants and was able to ameliorate PC12 cell ischaemic injury. PCB treatment significantly decreased brain infarct volume, limited the exploratory behaviour impairment and preserved viable cortical neurons in ischaemic rats in a dose-dependent manner, compared to the vehicle group. Furthermore, PCB at high doses restored the MBP and CNPase expression levels in ischaemic rats. An improved PCB purification method from its natural source is reported, obtaining PCB that is suitable for pharmacological trials showing neuroprotective effects against experimental ischaemic stroke. Therefore, PCB could be a therapeutic pharmacological alternative for ischaemic stroke patients.

Endothelin-1-mediated cerebral ischemia in mice: early cellular events and the role of caspase-3.

Over the past 30 years a number of animal models of cerebral ischemic injury have been developed. Middle cerebral artery occlusion (MCAO) in particular reproduces both ischemic and reperfusion elements and is widely utilized as a model of ischemic stroke in rodents. However substantial variability exists in this model even in clonal inbred mice due to stochastic elements of the cerebral vasculature. Models such as MCAO thus exhibit significant irreducible variabilities with respect to their zone of injury as well as inducing a sizable volume of injury to the cerebrum with damage to sub-cortical structures, conditions not typically seen for the majority of human clinical strokes. An alternative model utilizes endothelin-1 application focally to cerebral vasculature, resulting in an ischemic reperfusion injury which more closely mimics that seen in human clinical stroke. In order to further define this model we demonstrate that intra-cortical administration of ET-1 results in a highly reproducible pattern of tissue injury which is limited to the cerebral cortex, characterizing the early cellular and molecular events which occur during the first 24 h post-injury. In addition we demonstrate that caspase-3 is both necessary and sufficient to regulate a majority of cortical cell death observed during this period. The enhanced survival effects seen upon genetic deletion of caspase-3 appear to arise as a result of direct modification of cell autonomous PCD signaling as opposed to secondary effectors such as granulocyte infiltration or microglia activation. Taken together these findings detail the early mechanistic features regulating endothelin-1-mediated ischemic injury.

Neonatal cardiomyocyte hypertrophy induced by endothelin-1 is blocked by estradiol acting on GPER.

Estradiol (E2) prevents cardiac hypertrophy, and these protective actions are mediated by estrogen receptor (ER)α and ERß. The G protein-coupled estrogen receptor (GPER) mediates many estrogenic effects, and its activation in the heart has been observed in ischemia and reperfusion injury or hypertension models; however, the underlying mechanisms need to be fully elucidated. Herein, we investigated whether the protective effect of E2 against cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) is mediated by GPER and the signaling pathways involved. Isolated neonatal female rat cardiomyocytes were treated with ET-1 (100 nmol/l) for 48 h in the presence or absence of E2 (10 nmol/l) or GPER agonist G-1 (10 nmol/l) and GPER antagonist G-15 (10 nmol/l). ET-1 increased the surface area of cardiomyocytes, and this was associated with increased expression of atrial and brain natriuretic peptides. Additionally, ET-1 increased the phosphorylation of extracellular signal-related protein kinases-1/2 (ERK1/2). Notably, E2 or G-1 abolished the hypertrophic actions of ET-1, and that was reversed by G-15. Likewise, E2 reversed the ET-1-mediated increase of ERK1/2 phosphorylation as well as the decrease of phosphorylated Akt and its upstream activator 3-phosphoinositide-dependent protein kinase-1 (PDK1). These effects were inhibited by G-15, indicating that they are GPER dependent. Confirming the participation of GPER, siRNA silencing of GPER inhibited the antihypertrophic effect of E2. In conclusion, E2 plays a key role in antagonizing ET-1-induced hypertrophy in cultured neonatal cardiomyocytes through GPER signaling by a mechanism involving activation of the PDK1 pathway, which would prevent the increase of ERK1/2 activity and consequently the development of hypertrophy.

TRAF3IP2 mediates high glucose-induced endothelin-1 production as well as endothelin-1-induced inflammation in endothelial cells.

Hyperglycemia-induced production of endothelin (ET)-1 is a hallmark of endothelial dysfunction in diabetes. Although the detrimental vascular effects of increased ET-1 are well known, the molecular mechanisms regulating endothelial synthesis of ET-1 in the setting of diabetes remain largely unidentified. Here, we show that adapter molecule TRAF3 interacting protein 2 (TRAF3IP2) mediates high glucose-induced ET-1 production in endothelial cells and ET-1-mediated endothelial cell inflammation. Specifically, we found that high glucose upregulated TRAF3IP2 in human aortic endothelial cells, which subsequently led to activation of JNK and IKKß. shRNA-mediated silencing of TRAF3IP2, JNK1, or IKKß abrogated high-glucose-induced ET-converting enzyme 1 expression and ET-1 production. Likewise, overexpression of TRAF3IP2, in the absence of high glucose, led to activation of JNK and IKKß as well as increased ET-1 production. Furthermore, ET-1 transcriptionally upregulated TRAF3IP2, and this upregulation was prevented by pharmacological inhibition of ET-1 receptor B using BQ-788, or inhibition of NADPH oxidase-derived reactive oxygen species using gp91ds-tat and GKT137831. Notably, we found that knockdown of TRAF3IP2 abolished ET-1-induced proinflammatory and adhesion molecule (IL-1ß, TNF-α, monocyte chemoattractant protein 1, ICAM-1, VCAM-1, and E-selectin) expression and monocyte adhesion to endothelial cells. Finally, we report that TRAF3IP2 is upregulated and colocalized with CD31, an endothelial marker, in the aorta of diabetic mice. Collectively, findings from the present study identify endothelial TRAF3IP2 as a potential new therapeutic target to suppress ET-1 production and associated vascular complications in diabetes. NEW & NOTEWORTHY This study provides the first evidence that the adapter molecule TRAF3 interacting protein 2 mediates high glucose-induced production of endothelin-1 by endothelial cells as well as endothelin-1-mediated endothelial cell inflammation. The findings presented herein suggest that TRAF3 interacting protein 2 may be an important therapeutic target in diabetic vasculopathy characterized by excess endothelin-1 production.

Experience with the "good" limb induces aberrant synaptic plasticity in the perilesion cortex after stroke.

Following unilateral stroke, the contralateral (paretic) body side is often severely impaired, and individuals naturally learn to rely more on the nonparetic body side, which involves learning new skills with it. Such compensatory hyper-reliance on the "good" body side, however, can limit functional improvements of the paretic side. In rats, motor skill training with the nonparetic forelimb (NPT) following a unilateral infarct lessens the efficacy of rehabilitative training, and reduces neuronal activation in perilesion motor cortex. However, the underlying mechanisms remain unclear. In the present study, we investigated how forelimb movement representations and synaptic restructuring in perilesion motor cortex respond to NPT and their relationship with behavioral outcomes. Forelimb representations were diminished as a result of NPT, as revealed with intracortical microstimulation mapping. Using transmission electron microscopy and stereological analyses, we found that densities of axodendritic synapses, especially axo-spinous synapses, as well as multiple synaptic boutons were increased in the perilesion cortex by NPT. The synaptic density was negatively correlated with the functional outcome of the paretic limb, as revealed in reaching performance. Furthermore, in animals with NPT, there was dissociation between astrocytic morphological features and axo-spinous synaptic density in perilesion motor cortex, compared with controls. These findings demonstrate that skill learning with the nonparetic limb following unilateral brain damage results in aberrant synaptogenesis, potentially of transcallosal projections, and this seems to hamper the functionality of the perilesion motor cortex and the paretic forelimb.

Co-morbid beta-amyloid toxicity and stroke produce impairments in an ambiguous context task in rats without any impairment in spatial working memory.

Sporadic Alzheimer's disease (AD) accounts for a high proportion of AD cases. Therefore, it is of importance to investigate other factors that contribute to the etiology and progression of AD. AD is characterized by decreased cholinergic tone, tau hyperphosphorylation and beta-amyloid (Aß) accumulation. In addition to the hallmark pathology, other factors have been identified that increase the risk of AD, including stroke. This study examined the combined effects of beta-amyloid administration and unilateral stroke in an animal model of AD. Adult rats were given a sham surgery, bilateral intraventricular infusion of 10 µL of 50n mol Aß(25-35), a unilateral injection of endothelin-1 into the right striatum, or Aß and endothelin-1 administration in combination. Following a recovery period, rats were tested in the 1-trial place learning variant of the Morris water task followed by an ambiguous discriminative fear-conditioning to context task. After behavioural assessment, rats were euthanized, and representative sections of the medial septum were analyzed for differences in choline-acetyltransferase (ChAT) immunohistochemistry. No differences were observed in spatial working memory, but the combined effect of Aß and stroke resulted in deficits in the discriminative fear-conditioning to context task. A trend towards decreased ChAT-positive staining in the medial septum was observed. This study indicates that Aß and stroke in combination produce worse functional consequences than when experienced alone, furthering the concept of AD as a disease with multiple and complex etiologies.

Evidence for a role of the reticulospinal system in recovery of skilled reaching after cortical stroke: initial results from a model of ischemic cortical injury.

The purposes of this pilot study were to create a model of focal cortical ischemia in Macaca fascicularis and to explore contributions of the reticulospinal system in recovery of reaching. Endothelin-1 was used to create a focal lesion in the shoulder/elbow representation of left primary motor cortex (M1) of two adult female macaques. Repetitive microstimulation was used to map upper limb motor outputs from right and left cortical motor areas and from the pontomedullary reticular formation (PMRF). In subject 1 with a small lesion and spontaneous recovery, reaching was mildly impaired. Changes were evident in the shoulder/elbow representations of both the lesioned and contralesional M1, and there appeared to be fewer than expected upper limb responses from the left (ipsilesional) PMRF. In subject 2 with a substantial lesion, reaching was severely impaired immediately after the lesion. After 12 weeks of intensive rehabilitative training, reach performance recovered to near-baseline levels, but movement times remained about 50% slower. Surprisingly, the shoulder/elbow representation in the lesioned M1 remained completely absent after recovery, and there was a little change in the contralesional M1. There was a definite difference in motor output patterns for left versus right PMRF for this subject, with an increase in right arm responses from right PMRF and a paucity of left arm responses from left PMRF. The results are consistent with increased reliance on PMRF motor outputs for recovery of voluntary upper limb motor control after significant cortical ischemic injury.

Contralateral Hyperalgesia from Injection of Endothelin-1 into the Ipsilateral Paw Requires Efferent Conduction into the Contralateral Paw.

BACKGROUND: Contralateral hyperalgesia, occurring after unilateral injury, is usually explained by central sensitization in spinal cord and brain. We previously reported that injection of endothelin-1 (ET-1) into one rat hindpaw induces prolonged mechanical and chemical sensitization of the contralateral hindpaw. Here, we examined the role of contralateral efferent activity in this process. METHODS: ET-1 (2 nmol, 10 µL) was injected subcutaneously into the plantar surface of right (ipsilateral) hindpaw (ILP), and the thermal response latency and mechanical threshold for nocifensive withdrawal were determined by the use of, respectively, plantar radiant heating and von Frey filaments, for both ILP and contralateral hindpaws (CLP). Either paw was anesthetized for 60 minutes by direct injection of bupivacaine (0.25%, 40 µL), 30 minutes before ET-1. Alternatively, the contralateral sciatic nerve was blocked for 6 to 12 hours by percutaneous injection of bupivacaine-releasing microspheres 30 minutes before injection of ET-1. Systemic actions of these bupivacaine formulations were simulated by subcutaneous injection at the nuchal midline. RESULTS: After the injection of ET-1, the mechanical threshold of both ILP and CLP decreased by 2 hours, appeared to be lowest around 24 hours, and recovered through 48 hours to preinjection baseline at 72 hours. These hypersensitive responses were suppressed by bupivacaine injected into the ipsilateral paw before ET-1. Injection of the CLP by bupivacaine also suppressed the hypersensitivity of the CLP at all test times, and that of the ILP, except at 2 hours when it increased the sensitivity. This same pattern of change occurred when the contralateral sciatic nerve was blocked by bupivacaine-releasing microspheres. The systemic actions of these bupivacaine formulations were much smaller and only reached significance at 24 hours post-ET-1. Thermal hypersensitivity after ET-1 injection also occurred in both ILP and CLP and showed the same pattern in response to the 2 contralateral anesthetic procedures. CONCLUSIONS: These results show that efferent transmission through the contralateral innervation into the paw is necessary for contralateral sensitization by ET-1, suggesting that the release of substances by distal nerve endings is involved. The release of substances in the periphery is essential for contralateral sensitization by ET-1 and may also contribute to secondary hyperalgesia, occurring at loci distant from the primary injury, that occurs after surgery or nerve damage.

Epidermal growth factor and erythropoietin infusion accelerate functional recovery in combination with rehabilitation.

BACKGROUND AND PURPOSE: Rehabilitation is the only treatment option for chronic stroke deficits, but unfortunately, it often provides incomplete recovery. In this study, a novel combination of growth factor administration and rehabilitation therapy was used to facilitate functional recovery in a rat model of cortical stroke. METHODS: Ischemia was induced via injection of endothelin-1 into the sensorimotor cortex. This was followed by either a 2-week infusion of epidermal growth factor and erythropoietin or artificial cerebrospinal fluid into the ipsilateral lateral ventricle. Two weeks after ischemia, animals began an 8-week enriched rehabilitation program. Functional recovery was assessed after ischemia using the Montoya staircase-reaching task, beam-traversing, and cylinder test of forelimb asymmetry. RESULTS: The combination of growth factor infusion and rehabilitation led to a significant acceleration in recovery in the staircase task. When compared with controls, animals receiving the combination treatment attained significant recovery of function at 4 weeks after stroke, whereas those receiving rehabilitation alone did not recover until 10 weeks. Significant recovery was also observed on the beam-traversing and cylinder tasks. CONCLUSIONS: Combining behavioral rehabilitation with growth factor infusion accelerates motor recovery. These data suggest a promising new avenue of combination therapies that may have the potential to reduce the rehabilitation time necessary to recover from sensorimotor deficits arising from stroke.

Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing.

Stroke is the second cause of death worldwide with ischemic stroke accounting for 80% of all stroke insults. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events that lead to programmed cell death after traumatic brain injury. Alleviation of symptoms following ischemic neuronal injury can be potentially achieved by either genetic disruption or pharmacological inhibition of caspases. Here, we studied whether silencing of Caspase-3 using carbon nanotube-mediated in vivo RNA interference (RNAi) could offer a therapeutic opportunity against stroke. Effective delivery of siRNA directly to the CNS has been shown to normalize phenotypes in animal models of several neurological diseases. It is shown here that peri-lesional stereotactic administration of a Caspase-3 siRNA (siCas 3) delivered by functionalized carbon nanotubes (f-CNT) reduced neurodegeneration and promoted functional preservation before and after focal ischemic damage of the rodent motor cortex using an endothelin-1 induced stroke model. These observations illustrate the opportunity offered by carbon nanotube-mediated siRNA delivery and gene silencing of neuronal tissue applicable to a variety of different neuropathological conditions where intervention at well localized brain foci may offer therapeutic and functional benefits.