Nonischemic Animals Research Articles

In vivo effects of allogeneic mesenchymal stem cells in a rat model of acute ischemic kidney injury.

Objective(s):Renal ischemia-reperfusion injury (IRI) as a severe condition of acute kidney injury (AKI) is the most common clinical problem with high mortality rates of 35-60% deaths in hospital. Mesenchymal stem cells (MSC) due to unique regenerative characteristics are ideal candidates for the treatment of the ischemic injuries. This work is focused on the administration of MSC to IRI-induced AKI Wistar rats and evaluating their significance in AKI treatment. Material and Methods:Animals underwent surgical procedure and AKI was induced by 40 min bilateral renal pedicle clamping. Immediately after reperfusion, 2×106 rat bone marrow derived MSCs were injected via intra-parenchymal or intra-aortic route. Results:Animals subjected to AKI after days 1 and 3 showed significant increase in the serum creatinine and blood urea nitrogen (BUN) concentration along with a declined glomerular filtration rate (GFR) when compared with non-ischemic animals. On the other hand, treated animals showed a significant enhanced regeneration as compared to ischemic animals in both administration route groups. Conclusion:According to the results concluded from the renoprotective effects of MSC in IRI/AKI, MSCs could be considered as promising therapeutic approach for AKI in clinical applications.

A novel cell-penetrating peptide protects against neuron apoptosis after cerebral ischemia by inhibiting the nuclear translocation of annexin A1.

Nuclear translocation of annexin A1 (ANXA1) has recently been reported to participate in neuronal apoptosis after cerebral ischemia. Prevention of the nuclear translocation of ANXA1 should therefore inhibit neuronal apoptosis and protect against cerebral stroke. Here, we found that, in the repeat III domain of ANXA1, the amino-acid residues from R228 to F237 function as a unique nuclear translocation signal (NTS) and are required for nuclear translocation of ANXA1. Intriguingly, we synthesized a cell-penetrating peptide derived by conjugating the trans-activator of transcription (Tat) domain to the NTS sequence. This Tat-NTS peptide specifically blocked the interaction of ANXA1 with importin β and, consequently, the nuclear translocation of ANXA1 without affecting the nucleocytoplasmic shuttling of other proteins. The Tat-NTS peptide inhibited the transcriptional activity of p53, decreased Bid expression, suppressed activation of the caspase-3 apoptosis pathway and improved the survival of hippocampal neurons subjected to oxygen-glucose deprivation and reperfusion in vitro. Moreover, using a focal brain ischemia animal model, we showed that the Tat-NTS peptide could be efficiently infused into the ischemic hippocampus and cortex by unilateral intracerebroventricular injection. Injection of the Tat-NTS peptide alleviated neuronal apoptosis in the ischemic zone. Importantly, further work revealed that administration of the Tat-NTS peptide resulted in a dramatic reduction in infarct volume and that this was correlated with a parallel improvement in neurological function after reperfusion. Interestingly, the effects of Tat-NTS were injury specific, with little impact on neuronal apoptosis or cognitive function in sham-treated nonischemic animals. In conclusion, based on its profound neuroprotective and cognitive-preserving effects, it is suggested that the Tat-NTS peptide represents a novel and potentially promising new therapeutic candidate for the treatment of ischemic stroke.

Association of Long-Term Atorvastatin with Escalated Stroke-Induced Neuroinflammation in Rats.

Statins are widely used in high-risk patients to reduce the stroke incidence. However, little has been investigated about the impact of chronic pretreatment with statins on cerebral ischemic insult following defined arterial occlusion. To address this in experimental rats, in the present work, atorvastatin was orally dosed for 1month to evaluate the outcomes of the subsequent occlusive stroke induced by middle cerebral artery occlusion (MCAO). Our data was suggestive of potential escalating impact of chronic atorvastatin (Atv; 10mg/kg) on neurological function, but not infarct volume. According to our immunoblotting data, such escalations were consistent with the prominent rise in TNF-α and IL-6 which paralleled with augmented Bax/Bcl2 ratio and Caspase-9 activation; however, these were not enough to worsen acute neurodegeneration determined by Fluoro Jade B staining. Noteworthy, such deteriorating effects were also partly detected in non-ischemic animals. Conclusively, our data are indicative of cerebral proinflammatory effects of chronic Atv which might overwhelm the beneficial pliotropic of the drug and predispose animals' brain to ischemic insult. Further studies on different statins with discrete pharmacokinetic properties are highly suggested to precisely explore stroke outcomes following long term prophylactic treatment particularly in primates.

Abstract T P85: Mammalian Target of Rapamycin (mTOR) Signaling Pathway Mediates Cerebral Ischemic Brain Damage

BACKGROUND: Mammalian target of rapamycin (mTOR) signaling regulates cell energy metabolism and cell proliferation. Recent studies have demonstrated that mTOR activation is associated with increased cardiac infarct volume and necroptosis. The role of mTOR in cerebral ischemia has not been studied. The aim of this study is to explore how mTOR pathway behavior after cerebral ischemia and whether inhibition of mTOR reduces or increases ischemic brain damage. METHODS: Ten minutes of global ischemia was induced in Wistar rats (2-3 months old). Animals were either treatment with mTOR inhibitor rapamycin (6 mg/kg) or its vehicle. Animal brains were harvested after 3 hrs, 16 hrs and 7 days for histopathology and protein analyses. Sham-operated non-ischemic rats were used as control. Rapamycin was i.p. injected 1 hr after reperfusion in 7-day subgroups and injected 7 days prior to ischemia in 3 and 16 hrs subgroups. RESULTS: 10 minutes of global ischemia caused neuronal damage to the striatum, hippocampal CA1, and neocortical regions at 7 days of reperfusion. Levels of p-mTOR, p-S6K were increased significantly after 3 and 16 hrs in both the hippocampus and cortex. In addition, p-Akt and cytochrome c were elevated in the hippocampi after 3 hrs, peaked at 16 hrs and remained elevated after 7 days of reperfusion. Post-ischemic treatment with rapamycin 1 hrs after reperfusion significantly reduced brain damage in the CA1 and cortical areas at 7 days of reperfusion. Levels of p-mTOR, p-S6K, p-Akt and cytochrome c were significantly suppressed by rapamycin in non-ischemic and ischemic animals. CONCLUSION: Cerebral ischemia activates mTOR signaling pathway and inhibition of mTOR activativity reduces ischemic brain damage.

Brain-derived estrogen exerts anti-inflammatory and neuroprotective actions in the rat hippocampus

17β-estradiol (E2) has been implicated to play a critical role in neuroprotection, synaptic plasticity, and cognitive function. Classically, the role of gonadal-derived E2 in these events is well established, but the role of brain-derived E2 is less clear. To address this issue, we investigated the expression, localization, and modulation of aromatase and local E2 levels in the hippocampus following global cerebral ischemia (GCI) in adult ovariectomized rats. Immunohistochemistry (IHC) revealed that the hippocampal regions CA1, CA3 and dentate gyrus (DG) exhibited high levels of immunoreactive aromatase staining, with aromatase being co-localized primarily in neurons in non-ischemic animals. Following GCI, aromatase became highly expressed in GFAP-positive astrocytes in the hippocampal CA1 region at 2–3days post GCI reperfusion. An ELISA for E2 and IHC for E2 confirmed the GCI-induced elevation of local E2 in the CA1 region and that the increase in local E2 occurred in astrocytes. Furthermore, central administration of aromatase antisense (AS) oligonucleotides, but not missense (MS) oligonucleotides, blocked the increase in aromatase and local E2 in astrocytes after GCI, and resulted in a significant increase in GCI-induced hippocampal CA1 region neuronal cell death and neuroinflammation. As a whole, these results suggest that brain-derived E2 exerts important neuroprotective and anti-inflammatory actions in the hippocampal CA1 region following GCI.

English

The aim of the study is to investigate the effect of Echinacea on kidney and liver against oxidative stress during renal ischemia-reperfusion (I/R) injury, by determining biochemical parameters and evaluating histological examinations. Adult male Sprague-Dawley rats were evaluated in five groups (each group consists of 8 animals). Group 1 (Control; non-ischemic animals); Group 2 (renal I/R injury+saline), Group 3 (renal I/R injury + Echinacea, 25 mg/kg), Group 4 (renal I/R injury + Echinacea, 50 mg/kg) Group 5 (renal I/R injury+ Echinacea, 100 mg/kg) were designed to evaluate effects of Echinacea in renal I/R injury on the morphological changes in kidney and liver. A right nephrectomy was performed for I/R injury. On the 15th day following nephrectomy, ischemia and reperfusion was performed. Echinacea was administered intraperitoneally. Left kidney and liver were taken after 24 h of reperfusion period for histopathological examinations. The malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) enzyme activities were determined from tissue homogenates. In the study, Echinacea administration in renal ischemia-reperfusion decreased MDA in kidney and liver. SOD activity was decreased with low and high doses of Echinacea treatment in both kidney and liver. Echinacea administration decreased CAT levels in kidney and no difference was observed between Echinacea doses. CAT was increased in liver low dose group. But CAT was decreased in high doses of Echinacea. Histopathological examinations showed partial improvement in the damages seen in I/R injury after Echinacea administration at both kidney and liver. Echinacea may be effective in preventing oxidative injury.   Key words: Echinacea, ischemia-reperfusion, kidney, liver, antioxidant enzyme.

Targeted mini-strokes produce changes in interhemispheric sensory signal processing that are indicative of disinhibition within minutes

Most processing of sensation involves the cortical hemisphere opposite (contralateral) to the stimulated limb. Stroke patients can exhibit changes in the interhemispheric balance of sensory signal processing. It is unclear whether these changes are the result of poststroke rewiring and experience, or whether they could result from the immediate effect of circuit loss. We evaluated the effect of mini-strokes over short timescales (<2 h) where cortical rewiring is unlikely by monitoring sensory-evoked activity throughout much of both cortical hemispheres using voltage-sensitive dye imaging. Blockade of a single pial arteriole within the C57BL6J mouse forelimb somatosensory cortex reduced the response evoked by stimulation of the limb contralateral to the stroke. However, after stroke, the ipsilateral (uncrossed) forelimb response within the unaffected hemisphere was spared and became independent of the contralateral forelimb cortex. Within the unaffected hemisphere, mini-strokes in the opposite hemisphere significantly enhanced sensory responses produced by stimulation of either contralateral or ipsilateral pathways within 30-50 min of stroke onset. Stroke-induced enhancement of responses within the spared hemisphere was not reproduced by inhibition of either cortex or thalamus using pharmacological agents in nonischemic animals. I/LnJ acallosal mice showed similar rapid interhemispheric redistribution of sensory processing after stroke, suggesting that subcortical connections and not transcallosal projections were mediating the novel activation patterns. Thalamic inactivation before stroke prevented the bilateral rearrangement of sensory responses. These findings suggest that acute stroke, and not merely loss of activity, activates unique pathways that can rapidly redistribute function within the spared cortical hemisphere.

Effect of Hyperglycemia on Brain Penetrating Arterioles and Cerebral Blood Flow Before and After Ischemia/Reperfusion

The effect of preexisiting hyperglycemia on cerebral blood flow (CBF) and brain penetrating arterioles before and after 2 h of ischemia and 30 min of reperfusion was determined. Male Wistar rats that were either hyperglycemic (50 mg/kg streptozotocin; n=24) or normoglycemic (n=24) were subjected to transient ischemia by filament occlusion or nonischemic. CBF was measured prior to ischemia using microspheres and during transient ischemia using laser Doppler. Edema was compared by wet/dry weights. Constriction to apamin, TRAM-34, and L-NNA, inhibitors of small- and intermediate-conductance calcium-activated potassium channels (SK and IK) and nitric oxide, were compared in penetrating arterioles from the ischemic hemisphere to investigate changes in basal tone and endothelium-dependent vasodilator responses. Preexisiting hyperglycemia did not affect CBF in non-ischemic animals or after transient ischemia; however, edema was significantly greater. Ischemia and reperfusion caused decreased basal tone in penetrating arterioles similarly in normoglycemic and hyperglycemic animals that was restored by apamin, and further increased by TRAM-34 and L-NNA. The restoration of tone in penetrating arterioles by apamin and TRAM-34 suggests that transient ischemia activates SK and IK channels in penetrating arterioles. This effect of ischemia was not different between normoglycemic and hyperglycemic animals, suggesting that it was related to ischemia and reperfusion rather than hyperglycemia.

Fenofibrate Improves Cerebral Blood Flow after Middle Cerebral Artery Occlusion in Mice

Fibrates, one group of peroxisome proliferator-activated receptor (PPAR) activators, are lipid lowering drugs. Fibrates have been shown to attenuate brain tissue injury after focal cerebral ischemia. In this study, we investigated the impact of fenofibrate on cerebral blood flow (CBF) in male wild type and PPARalpha-null mice. Animals were treated for 7 days with fenofibrate and subjected to 2 h of filamentous middle cerebral artery occlusion and reperfusion under isoflurane anesthesia. Cortical surface CBF was measured by laser speckle imaging. Regional CBF (rCBF) in nonischemic animals was measured by (14)C-iodoantipyrine autoradiography. Fenofibrate did not affect rCBF and mean arterial blood pressure in nonischemic animals. In ischemic animals, laser speckle imaging showed delayed expansions of ischemic area, which was attenuated by fenofibrate. Fenofibrate also enhanced CBF recovery after reperfusion. However, such effects of fenofibrate on CBF in the ischemic brain were not observed in PPARalpha-null mice. These findings show that fenofibrate improves CBF in the ischemic hemisphere. Moreover, fenofibrate requires PPARalpha expression for the cerebrovascular protective effects in the ischemic brain.

Expression of MMP-12 after Neonatal Hypoxic-Ischemic Brain Injury in Mice

Matrix metalloproteinase-12 (MMP-12) is expressed in the brain and is important for myelin formation in the developing brain, while MMP-12 deficiency protects against spinal cord injury in the adult, suggesting a role for MMP-12 after brain injury. However, the role of MMP-12 in neonatal hypoxic-ischemic brain injury is not known. The purpose of this study was to investigate the expression of MMP-12 in the brain after neonatal hypoxia-ischemia (HI). HI was induced by unilateral ligation of the left carotid artery followed by hypoxia (10% O₂, 36°C) for 50 min in postnatal day 9 C57/Bl6J mice. At 24 and 72 h after HI, the mRNA and protein expression of MMP-12 were measured by real-time PCR and Western blot, respectively. Distribution and cellular expression of MMP-12 were examined by immunohistochemistry. At 72 h after HI, both MMP-12 mRNA and protein expression were significantly increased in the ipsilateral hemisphere compared to the contralateral hemisphere and sham-operated animals (p < 0.05). The extent of tissue loss in the ipsilateral hemisphere at 72 h after HI was positively correlated with the degree of MMP-12 protein expression (r = 0.900, p < 0.05). In nonischemic animals, immunohistochemical analysis demonstrated weak cytoplasmic MMP-12 immunoreactivity throughout the brain in cells which resembled neurons and stronger immunoreactivity was observed in blood vessels. After HI, the MMP-12 staining became more prominent in the ipsilateral hemisphere and immunohistochemical double-labeling experiments identified the MMP-12-positive cells as neurons, isolectin and Olig2-positive cells. This study demonstrates that MMP-12 is upregulated after HI, suggesting that MMP-12 may contribute to injury in the immature brain, similar to that seen in the adult.

Dose‐dependent effect of S(+) ketamine on post‐ischemic endogenous neurogenesis in rats

Ketamine is a non-competitive antagonist at N-methyl-D-aspartate (NMDA) receptors and reduces neuronal injury after cerebral ischemia by blocking the excitotoxic effects of glutamate. However, cerebral regeneration by means of endogenous neurogenesis may be impaired with blockade of NMDA receptors. The effects of S(+) ketamine on post-ischemic neurogenesis are unknown and investigated in this study. Thirty-two male Sprague-Dawley rats were randomly assigned to the following treatment groups with intravenous S(+) ketamine anesthesia: S(+) ketamine 0.75 mg/kg/min with or without cerebral ischemia and S(+) ketamine 1.0 mg/kg/min with or without cerebral ischemia. Eight non-anesthetized, non-ischemic animals were investigated as naïve controls. Forebrain ischemia was induced by bilateral common carotid artery occlusion in combination with hemorrhagic hypotension. 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally for seven consecutive post-operative days. BrdU-positive neurons in the dentate gyrus and histopathological damage of the hippocampus were analyzed after 28 days. The number of new neurons was not affected by S(+) ketamine in the absence of cerebral ischemia. The ischemia-induced increase in neurogenesis was reduced by high-dose S(+) ketamine. Cell death of ischemic animals did not vary between low- and high-dose S(+) ketamine. While low concentrations of S(+) ketamine allow an ischemia-induced increase in the number of new neurons, high S(+) ketamine concentrations block the post-ischemic increase in newly generated neurons. This effect is irrespective of the extent of other histopathological damage and in line with studies showing that NMDA receptor antagonists like MK-801 inhibit neurogenesis after cerebral ischemia.

Cerebral Ischemia and Neurogenesis: A Two-time Comparison

This study compares the effect of mild and severe cerebral ischemia on neuronal damage and neurogenesis. Sixteen Sprague-Dawley rats, anesthetized with 0.8 vol% halothane in O(2)/air, were subjected to forebrain ischemia by bilateral common carotid artery occlusion plus hemorrhagic hypotension (mean arterial blood pressure = 40 mmHg) for 8 (mild) or 13 (severe) min. Four non-ischemic animals were investigated as naïve controls. Bromodeoxyuridine (50 mg/kg), a marker of new cells, was administrated for seven consecutive postischemic days. After 28 days, animals were perfused with 4% paraformaldehyde and the brains were sliced. Histopathological damage of the hippocampus and the volume of the dentate gyrus were assessed by HE-staining. With immunohistochemistry BrdU-positve cells were detected in the dentate gyrus. The amount of new generated neurons was identified by double-immunofluorescence-staining of BrdU and neuronal marker (NeuN). In the CA-1 region of the hippocampus, mild ischemia induced damage up to 10% (HE-index 0.8 +/- 1.2) and severe ischemia up to 50% (HE-index 2.1 +/- 1.4). There was no histopathological damage in naïve control animals. The amount of new neurons was increased by 250% after mild insult and by 160% after severe insult compared to the naïve control animals. These data indicate that histopathological damage depends on the severity of the ischemic insult and that forebrain ischemia activates generation of new neurons. A mild ischemic challenge appears to be a more potent neurogenic stimulus than severe ischemia. The new neurons survive at least 28 days. This may relate to delayed histopathological and functional recovery after cerebral ischemia.

Melatonin Protects Myocardium from Ischemia-Reperfusion Injury in Hypertensive Rats: Role of Myeloperoxidase Activity

Increased levels of reactive oxygen species, alterations in nitric oxide synthesis, and increased migration of neutrophils to the ischemic tissue play an important role in the pathophysiology of myocardial ischemia-reperfusion (IR) injury. In this study, we have evaluated the effects of melatonin on myeloperoxidase (MPO) activity, tissue glutathione (GSH), lipid peroxidation levels, and blood pressure in L-NAME-induced hypertensive rats with or without IR. NOS inhibitor L-NAME was administrated before inducing cardiac ischemia for 15 days intraperitoneally. For the cardiac ischemia, the left coronary artery was ligated for 30 min, and reperfusion was performed for 120 min after the ischemia. L-NAME treatment in non-ischemic animals increased blood pressure and lipid peroxidation, and decreased glutathione level in myocardial tissue significantly as compared with non-L-NAME-treated animals. Melatonin reversed L-NAME-induced blood pressure elevation and oxidative changes. Cardiac IR increased MDA levels and MPO activity and decreased GSH levels as compared with non-ischemic animals. L-NAME treatment did not change in IR-induced MDA and GSH levels as compared with ischemic control animals. However, MPO activity was significantly higher than control ischemic animals. MDA levels and MPO activity resulting from ischemic injury in melatonin-treated animals were significantly less than L-NAME-treated animals. Taken together—the ischemic and non-ischemic control and melatonin-treated animals—this study shows that neutrophil migration plays an important role on the development of ischemic injury in hypertensive rats.

Contribution of Poly(ADP-Ribose) Polymerase to Postischemic Blood—Brain Barrier Damage in Rats

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays a significant role in postischemic brain injury. We assessed the contribution of PARP activation to the blood-brain barrier (BBB) disruption and edema formation after ischemia-reperfusion. In male Wistar rats, global cerebral ischemia was achieved by occluding the carotid arteries and lowering arterial blood pressure for 20 mins. The animals were treated with saline or with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl (PJ34); (10 mg/kg, i.v.) before ischemia. After 40 mins, 24, and 48 h of reperfusion, the permeability of the cortical BBB was determined after Evans Blue (EB) and Na-fluorescein (NaF) administration. The water content of the brain was also measured. The permeability of the BBB for EB increased after ischemia-reperfusion compared with the nonischemic animals after 24 and 48 h reperfusion but PARP inhibition attenuated this increase at 48 h (nonischemic: 170+/-9, saline: 760+/-95, PJ34: 472+/-61 ng/mg tissue). The extravasation of NaF showed similar changes and PJ34 post-treatment attenuated the permeability increase even at 24 h. PARP inhibition decreased the brain edema seen at 48 h. Because PARP has proinflammatory properties, the neutrophil infiltration of the cortex was determined, which showed lower values after PJ34 treatment. Furthermore, PJ34 treatment decreased the loss of the tight junction protein occludin at 24 and 48 h. The inhibition of PARP activity accompanied by reduced post-ischemic BBB disturbance and decreased edema formation suggests a significant role of this enzyme in the development of cerebral vascular malfunction

Peroxynitrite Diminishes Myogenic Activity and Is Associated With Decreased Vascular Smooth Muscle F-Actin in Rat Posterior Cerebral Arteries

This study investigated the effect of peroxynitrite (ONOO-) on pressure-induced myogenic activity and vascular smooth muscle (VSM) actin of isolated posterior cerebral arteries (PCAs). Histochemical staining of nitrotyrosine (NT) was used to demonstrate the presence of ONOO- in the cerebrovasculature after 1 hour of middle cerebral artery occlusion with 30 minutes of reperfusion. To determine the effect of ONOO- on pressure-induced myogenic activity, third-order PCAs from nonischemic animals were isolated and mounted in an arteriograph chamber. Diameter in response to changes in pressure was determined in the absence and presence of ONOO- (10(-8) to 10(-4) mol/L). Filamentous actin (F-actin) and globular actin (G-actin) were quantified using confocal microscopy in PCAs with and without exposure to ONOO-. NT staining of vascular cells was greater in ischemic brain versus sham animals (56+/-3% versus 35+/-3%; P<0.01). Addition of low concentrations of ONOO- (< or =10(-6) mol/L) to isolated PCAs caused constriction from 129+/-16 microm to 115+/-15 microm (P<0.01), whereas concentrations >10(-6) mol/L caused dilation of spontaneous tone and loss of myogenic activity in the physiological range of 50 to 125 mm Hg, increasing diameter from 130+/-6 to 201+/-5 microm at 75 mm Hg (P<0.01). In addition, the diminished myogenic activity was associated with a 4.5-fold decrease in F-actin content of VSM and a 27% increase in G-actin content (P<0.01). This study demonstrates that ONOO- affects the myogenic activity of cerebral arteries and causes F-actin depolymerization in VSM, a consequence that could promote vascular damage during reperfusion injury and further brain injury.

917. GM-CSF Gene Transfer to Acute Limb Ischemic Mouse for Limb Recuperation

IntroductionPeripheral arterial diseases may be caused by any occlusive leg artery lesion that interferes with blood flow. When these conditions occur, there is an increase in vessel resistance that can lead to a reduction in distal perfusion pressure and blood flow. This process forces collateral vessel remodeling and it is known as arteriogenesis. Inflammation is the main triggering factor of arteriogenesis and several molecular factors are involved. One way to prolong the arteriogenic activity is extending monocytes half-life by apoptosis inhibition with GM-CSF (granulocyte and macrophage colony stimulating factor). This factor is also responsible for proliferation and differentiation of inflammatory cells, which are important growth factors producers and chemoattractants for arteriogenesis.The main objective of this study was to assess the capacity of reverting surgery-induced acute limb ischemia in mice by in vivo transference of plasmid vector with GM-CSF gene and to compare with the VEGF gene transfer.Materials and MethodsTwo plasmid vectors were used in this study: uP-GM and uP- VEGF to express GM-CSF and VEGF, respectively. To induce acute limb ischemia in mice BALB/c, the femoral artery was excised from its origin of the external iliac artery to the distal point, where it bifurcates into saphenous and popliteal arteries, without damaging the vein and femoral nerve. All branches of femoral artery were ligated. Gene therapy was performed by injecting 100 μg of plasmid vector at the thigh soon after the ischemia surgery.Results and DiscussionAbout 30 % of animals that underwent ischemic surgery lost their limbs after 1 week, meanwhile none of uP-GM treated animals lost their limbs, even after 4 weeks of the surgery. Functional tests of the GM-CSF gene treated muscles revealed about 70 % of recovery of force, meanwhile the VEGF gene treated animals and the non- treated animals recovered only 25 % and 5 % of force respectively after 4 weeks of the gene transfer. Weight of gastrocnemius reached 100 % of recovery with ischemic animals treated with uP-GM after 4 weeks in comparison to the non-ischemic animals, but the VEGF gene treated or non-treated animals showed recovery of only 60 %. Capillary density had a significant increase in the VEGF treated animals after 1 week of surgery, but those treated with GM-CSF did not change in comparison to the non-treated ischemic animals, and this profile continued for 4 weeks. Histological data from the animals treated with GM-CSF for 4 weeks revealed that the muscle organization returned to the normal form.Therefore, we conclude that GM-CSF gene transference therapy in the ischemic muscle promoted good recovery of physical and physiological parameters in the long term meanwhile the use of VEGF gene promoted rapid recovery but it was not sustainable for 4 weeks.

TRANSENDOCARDIAL AND TRANSEPICARDIAL INTRAMYOCARDIAL FIBROBLAST GROWTH FACTOR-2 ADMINISTRATION: MYOCARDIAL AND TISSUE DISTRIBUTION

Effective local delivery to the heart remains an obstacle to successful therapeutic application of a number of drugs and biological agents. This study was designed to study and optimize the delivery characteristics of transendocardial intramyocardial (IM) administration, determine myocardial deposition and retention over time, and compare it to transepicardial IM injection. Thirty-nine pigs were used for the study (15 for catheter optimization, 15 for transendocardial IM delivery, and 9 for transepicardial IM delivery). (125)I-Fibroblast growth factor-2 (FGF2) (25 microCi) was used as the prototype molecule. Tissue and myocardial distribution was determined at 1 and 24 h and 7 days. Using 1-h (125)I-FGF2 myocardial deposition as a parameter for delivery efficiency, the optimal needle length and delivery volume for transendocardial based delivery were determined to be 6 mm and 0.1 ml, respectively. Using these parameters for endocardial delivery, (125)I-FGF2 cardiac activity was 18.01 +/- 3.84% of delivered activity at 1 h, 11.65 +/- 5.17% at 24 h, and 2.32 +/- 0.87% at 7 days in ischemic animals. Studies in nonischemic animals produced similar results. For transepicardial delivery, (125)I-FGF2 cardiac-specific activity was 23.14 +/- 12.67% for the 6-mm needle, declining to 12.32 +/- 8.50% at 24 h, and did not significantly differ from values obtained following transendocardial delivery. Thus, optimized transendocardial intramyocardial delivery using Biosense guidance results in efficient delivery of FGF2 to the target myocardium that is comparable with transepicardial delivery, both providing markedly higher myocardial deposition and retention and lower systemic recirculation of FGF2 than intracoronary, intrapericardial, or intravenous delivery. However, myocardial distribution is limited to injection sites.

Changes in non-heme iron histochemistry in the ischemic brains of cats and Mongolian gerbils

We studied the changes in non-heme iron histochemistry in the ischemic brains of the cat and Mongolian gerbils. For the histochemical visualization of non-heme ferric (Fe(III)) and ferrous (Fe(II)) iron, perfusion-Perls and perfusion-Turnbull methods were used. Deeply anesthetized animals were transcardially perfused with the solution containing 1% HCl, 1% potassium ferrocyanide (perfusion-Perls method) or 1% potassium ferricyanide (perfusion-Turnbull method), and 10% formalin. In the non-ischemic animals, perfusion was performed in the cephalic direction through the abdominal aorta or through the ascending aorta within 2 min after thorachotomy and cardiotomy—incision of the right auricle and left ventricle. In the ischemic animals, perfusion was performed through the ascending aorta 10, 20 and 30 min after cardiotomy. Frozen sections of the brain were treated for 3,3′-diaminobenzidine tetrahydrochloride (DAB) intensification of Perls and Turnbull reaction product. It was demonstrated that cytoplasmic Fe(III) was reduced to Fe(II) in oligodendrocytes of ischemic brains, and that Fe(II) was concentrated in the neuronal and glial cell nuclei regardless of the presence or absence of blood supply impairment.

Effects of hypothermia on neuronal-vascular function after cerebral ischemia in piglets.

We determined whether cerebral arteriolar dilation to N-methyl-d-aspartate (NMDA), a response dependent on stimulation of cortical neurons and inhibited by anoxic stress, would be preserved by hypothermia during and following ischemia. Pial arteriolar diameters in anesthetized piglets were determined via intravital microscopy. Arteriolar responses to NMDA (10, 50, and 100 micromol/l) were measured before and 1 h after 10 min of global ischemia. Piglets were exposed to either total body or selective brain cooling (33-34 degrees C). Arteriolar dilation to lower doses or to 100 micromol/l NMDA was not affected by hypothermia alone (51 +/- 3 vs. 46 +/- 7%, normothermia vs. hypothermia; n = 7) in nonischemic animals. However, arteriolar responses to 100 micromol/l NMDA were clearly attenuated after ischemia despite body cooling during ischemia (53 +/- 3 vs. 32 +/- 6%; n = 8), hypothermia during ischemia and early reperfusion (49 +/- 10 vs. 20 +/- 3%; n = 8), or selective brain cooling (48 +/- 5 vs. 20 +/- 5%; n = 10). In contrast, pretreatment with indomethacin resulted in complete preservation of NMDA-induced vasodilation after ischemia. Thus, hypothermia fails to protect against neuronal dysfunction during ischemia.

Electrical responses in hippocampal slices after prolonged global ischemia: effects of neuroprotectors

A simple and reproducible animal model of global ischemia, induced by decapitation in 30-day-old Wistar rats, has been developed. It allows to perform electrophysiological analysis of the postischemic reperfusion period in the brain slices. Periods of ischemia up to 40 min increase population spikes measured in the CA1 area of the hippocampus during 2–5 h of reperfusion. Thus after 30-min decapitation-induced ischemia (at t ischem=25°C), the mean amplitude of the recorded maximum orthodromic population spikes was 159% of the control obtained in the non-ischemic animals. Longer ischemic episodes result in the depression of the population spikes. After 2 h of ischemia, the amplitude of population spikes was about 89% of control. After 3 h of decapitation ischemia, the neurons could not be reactivated. The duration of ischemic episode needed for the irreversible depression of the electrical activity of the brain neurons drastically depends on the temperature at which the ischemic brain is maintained. Thus, only 2 h were needed at 30°C as compared to nearly 3 h at 25°C. We have found that intraperitoneal injection of neuroprotectors which precedes decapitation enables reactivation of the post-ischemic neurons even after very long periods of global ischemia. Thus, MK-801, a non-competitive NMDA receptors antagonist, or NBQX, a blocker of AMPA receptors, administrated 15 min before the long-term (90 min) decapitation ischemia (30°C), induced dose-dependent recovery of population spike with ED 50 values 0.2 mg/kg and 3 mg/kg respectively. Our results demonstrate that, in spite of the high vulnerability of hippocampal neurons to hypoxia and ischemia, their electrical activity can be restored after prolonged (more then 1 h) decapitation ischemia. Administration of NMDA or AMPA antagonists enhances recovery.