The Mn porphyrins of k(cat)(O(2)(.-)) as high as that of a superoxide dismutase enzyme, and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP(5+) is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP(5+) has been in part attributed to its micellar properties, i.e. the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH(2) group by oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP(5+). The high SOD-like activity is fully preserved and somewhat enhanced: log k(cat)(O(2)(.-))=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP(5+) and MnTnHep-2-PyP(5+), respectively. MnTnBuOE-2-PyP(5+) was tested in an O(2)(.-)- specific in vivo assay - aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5-30μM. MnTnHep-2-PyP(5+) was already toxic at 5μM, and MnTnHex-2-PyP(5+) became toxic at 30μM. In a mouse toxicity study, MnTnBuOE-2-PyP(5+) was several-fold less toxic than either MnTnHex-2-PyP(5+) or MnTnHep-2-PyP(5+).

Traumatic brain injury (TBI) and intracerebral hemorrhage (ICH) are leading causes of neurological mortality and disability in the US. However, therapeutic options are limited and clinical management remains largely supportive. HMG CoA reductase inhibitors (statins) have pleiotropic mechanisms of action in the setting of acute brain injury, and have been demonstrated to improve outcomes in preclinical models of ICH and TBI. To facilitate translation to clinical practice, we now characterize the optimal statin and dosing paradigm in murine models of ICH and TBI. In a preclinical model of TBI, mice received vehicle, simvastatin, and rosuvastatin at doses 1mg/kg and 5mg/kg for 5 days after the impact. Immunohistochemistry, differential gene expression, and functional outcomes (rotarod and Morris water maze) were assessed to gauge treatment response. Following TBI, administration of rosuvastatin 1 mg/kg was associated with the greatest improvement in functional outcomes. Rosuvastatin treatment was associated with histological evidence of reduced neuronal degeneration at 24 hours post-TBI, reduced microgliosis at day 7 post-TBI, and preserved neuronal density in the CA3 region at 35 days post-injury. Administration of rosuvastatin following TBI was also associated with downregulation of inflammatory gene expression in brain. Following ICH, treatment with simvastatin 1mg/kg was associated with the greatest improvement in functional outcomes, an effect that was independent of hemorrhage volume. Clinically relevant models of acute brain injury may be used to define variables such as optimal statin and dosing paradigms to facilitate the rational design of pilot clinical trials.

Intracerebroventricular treatment with redox-regulating Mn(III) N-hexylpyridylporphyrin (MnPorphyrin) is remarkably efficacious in experimental central nervous system (CNS) injury. Clinical development has been arrested because of poor blood-brain barrier penetration. Mn(III) meso-tetrakis (N-hexylpyridinium-2-yl) porphyrin (MnTnHex-2-PyP) was synthesized to include four six-carbon (hexyl) side chains on the core MnPorphyrin structure. This has been shown to increase in vitro lipophilicity 13,500-fold relative to the hydrophilic ethyl analog Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). In normal mice, we found brain MnTnHex-2-PyP accumulation to be ∼9-fold greater than MnTE-2-PyP 24 h after a single intraperitoneal dose. We then evaluated MnTnHex-2-PyP efficacy in outcome-oriented models of focal cerebral ischemia and subarachnoid hemorrhage. For focal ischemia, rats underwent 90-min middle cerebral artery occlusion. Parenteral MnTnHex-2-PyP treatment began 5 min or 6 h after reperfusion onset and continued for 7 days. Neurologic function was improved with both early (P = 0.002) and delayed (P = 0.002) treatment onset. Total infarct size was decreased with both early (P = 0.03) and delayed (P = 0.01) treatment. MnTnHex-2-PyP attenuated nuclear factor κB nuclear DNA binding activity and suppressed tumor necrosis factor-α and interleukin-6 expression. For subarachnoid hemorrhage, mice underwent perforation of the anterior cerebral artery and were treated with intraperitoneal MnTnHex-2-PyP or vehicle for 3 days. Neurologic function was improved (P = 0.02), and vasoconstriction of the anterior cerebral (P = 0.0005), middle cerebral (P = 0.003), and internal carotid (P = 0.015) arteries was decreased by MnTnHex-2-PyP. Side-chain elongation preserved MnPorphyrin redox activity, but improved CNS bioavailability sufficient to cause improved outcome from acute CNS injury, despite delay in parenteral treatment onset of up to 6 h. This advance now allows consideration of MnPorphyrins for treatment of cerebrovascular disease.

Cationic Mn(III) N-alkylpyridylporphyrins (MnPs) are potent SOD mimics and peroxynitrite scavengers and diminish oxidative stress in a variety of animal models of central nervous system (CNS) injuries, cancer, radiation, diabetes, etc. Recently, properties other than antioxidant potency, such as lipophilicity, size, shape, and bulkiness, which influence the bioavailability and the toxicity of MnPs, have been addressed as they affect their in vivo efficacy and therapeutic utility. Porphyrin bearing longer alkyl substituents at pyridyl ring, MnTnHex-2-PyP(5+), is more lipophilic, thus more efficacious in vivo, particularly in CNS injuries, than the shorter alkyl-chained analog, MnTE-2-PyP(5+). Its enhanced lipophilicity allows it to accumulate in mitochondria (relative to cytosol) and to cross the blood-brain barrier to a much higher extent than MnTE-2-PyP(5+). Mn(III) N-alkylpyridylporphyrins of longer alkyl chains, however, bear micellar character, and when used at higher levels, become toxic. Recently we showed that meta isomers are ∼10-fold more lipophilic than ortho species, which enhances their cellular accumulation, and thus reportedly compensates for their somewhat inferior SOD-like activity. Herein, we modified the alkyl chains of the lipophilic meta compound, MnTnHex-3-PyP(5+) via introduction of a methoxy group, to diminish its toxicity (and/or enhance its efficacy), while maintaining high SOD-like activity and lipophilicity. We compared the lipophilic Mn(III) meso-tetrakis(N-(6'-methoxyhexyl)pyridinium-3-yl)porphyrin, MnTMOHex-3-PyP(5+), to a hydrophilic Mn(III) meso-tetrakis(N-(2'-methoxyethyl)pyridinium-3-yl)porphyrin, MnTMOE-3-PyP(5+). The compounds were characterized by uv-vis spectroscopy, mass spectrometry, elemental analysis, electrochemistry, and ability to dismute O(2)˙(-). Also, the lipophilicity was characterized by thin-layer chromatographic retention factor, R(f). The SOD-like activities and metal-centered reduction potentials for the Mn(III)P/Mn(II)P redox couple were similar-to-identical to those of N-alkylpyridyl analogs: log k(cat)= 6.78, and E(1/2)=+68 mV vs. NHE (MnTMOHex-3-PyP(5+)), and log k(cat)= 6.72, and E(1/2)=+64 mV vs. NHE (MnTMOE-3-PyP(5+)). The compounds were tested in a superoxide-specific in vivo model: aerobic growth of SOD-deficient E. coli, JI132. Both MnTMOHex-3-PyP(5+) and MnTMOE-3-PyP(5+) were more efficacious than their alkyl analogs. MnTMOE-3-PyP(5+) is further significantly more efficacious than the most explored compound in vivo, MnTE-2-PyP(5+). Such a beneficial effect of MnTMOE-3-PyP(5+) on diminished toxicity, improved efficacy and transport across the cell wall may originate from the favorable interplay of the size, length of pyridyl substituents, rotational flexibility (the ortho isomer, MnTE-2-PyP(5+), is more rigid, while MnTMOE-3-PyP(5+) is a more flexible meta isomer), bulkiness and presence of oxygen.

BACKGROUND AND PURPOSE S-nitrosylated hemoglobin (S-nitrosohemoglobin) has been implicated in the delivery of O(2) to tissues through the regulation of microvascular blood flow. This study tested the hypothesis that enhancement of S-nitrosylated hemoglobin by ethyl nitrite inhalation improves outcome after experimental subarachnoid hemorrhage (SAH). METHODS A preliminary dosing study identified 20 ppm ethyl nitrite as a concentration that produced a 4-fold increase in S-nitrosylated hemoglobin concentration with no increase in methemoglobin. Mice were subjected to endovascular perforation of the right anterior cerebral artery and were treated with 20 ppm ethyl nitrite in air, or air alone for 72 hours, after which neurologic function, cerebral vessel diameter, brain water content, cortical tissue Po(2), and parenchymal red blood cell flow velocity were measured. RESULTS At 72 hours after hemorrhage, air- and ethyl nitrite-exposed mice had similarly sized blood clots. Ethyl nitrite improved neurologic score and rotarod performance; abated SAH-induced constrictions in the ipsilateral anterior, middle cerebral, and internal carotid arteries; and prevented an increase in ipsilateral brain water content. Ethyl nitrite inhalation increased red blood cell flow velocity and cortical tissue Po(2) in the ipsilateral cortex with no effect on systemic blood pressure. CONCLUSIONS Targeted S-nitrosylation of hemoglobin improved outcome parameters, including vessel diameter, tissue blood flow, cortical tissue Po(2), and neurologic function in a murine SAH model. Augmenting endogenous Po(2)-dependent delivery of NO bioactivity to selectively dilate the compromised cerebral vasculature has significant clinical potential in the treatment of SAH.

BRIEF SUMMARY: We describe the use of adenosine-induced cardiac arrest to facilitate intracranial aneurysm clip ligation. Cerebral aneurysms are highly variable which may result in difficult surgical exposure for clip ligation in select cases. Secure clip placement is often not feasible without temporarily decompressing the aneurysm. This can be accomplished with temporary clip ligation of proximal vessels, or with deep hypothermic circulatory arrest on cardiopulmonary bypass, although these methods have their own inherent risks. Here we describe an alternate method of decompressing the aneurysm via adenosine-induced transient asystole. We examined the records of 27 patients who underwent craniotomy for cerebral aneurysm clipping in which adenosine was used to induce transient asystole to facilitate clip ligation. Duration of adenosine-induced bradycardia (heart rate <40) and hypotension (SBP < 60) recorded on the electronic anesthesia record and outcome data including incidence of successful clipping, intraoperative and postoperative complications, and mortality were recorded. Satisfactory aneurysm decompression was achieved in all cases, and all aneurysms were clipped successfully. The median dose of intravenous adenosine resulting in bradycardia greater than 30 seconds was 30 mg. The median dose of adenosine resulting in hypotension greater than 30 seconds was 15 mg, and greater than 60 seconds was 30 mg. One case of prolonged hypotension after rapid redosing of adenosine required brief closed chest compressions before circulation was spontaneously restored. No other adverse events were observed. Adenosine cardiac arrest is a relatively novel method for decompression of intracranial aneurysms to facilitate clip application. With appropriate safety precautions, it is a reasonable alternative method when temporary clipping of proximal vessels is not desirable or not possible.

Use of genetically manipulated mice facilitates understanding pathological mechanisms in many diseases and contributes to therapy development. However, there is no practical and clinically relevant mouse model available for spinal cord ischemia. This report introduces a simplified long-term outcome mouse model of spinal cord ischemia. Male C57Bl/6J mice were anesthetized with isoflurane and endotracheally intubated. The middle segment of the thoracic aorta was clamped for 0, 8, 10 or 12min via left lateral thoracotomy. Rectal temperature was maintained at 37.0+/-0.5 degrees C. A laser Doppler probe was used to measure lumbar spinal cord blood flow during thoracic aorta cross-clamping. Open field locomotor function and rotarod performance were evaluated at 1h and 1, 3, 5, and 7 days postinjury. Surviving neurons in the lumbar ventral horn were counted at 7 days post-injury. Crossclamping the middle segment of the thoracic aorta resulted in approximately 90% blood flow reduction in the lumbar spinal cord. Neurological deficit and neuronal cell death were associated with ischemia duration. Another set of mice were subjected to 10min aortic clamping or sham surgery and neurological function was examined at 1 hour and 1, 3, 5, 7, 14, and 28 days. Four of 5 mice (80%) in the injured group survived 28 days and had significant neurological deficit. This study indicates that cross-clamping of the aorta via left thoracotomy is a simple and reliable method to induce spinal cord ischemia in mice allowing definition of long-term outcome.

Purpose: We examined the effects of manganese (III) meso-tetrakis (diethyl-2-5-imidazole) porphyrin, a metalloporphyrin antioxidant (MPA), on neural tissue radiation toxicity in vivo and on tumour cell radiosensitivity in vitro. Materials and methods: MPA was administered directly into the right lateral ventricle of young adult, male Sprague-Dawley rats (0 or 3.4 mug) 3 h before treatment with a single fraction, 100 Gy radiation dose delivered to the left brain hemisphere. The effects of treatment on radiation responses were assessed at different time points following irradiation. Results: MPA treatment prior to brain irradiation protected against acute radiation-induced apoptosis and ameliorated delayed damage to the blood-brain barrier and radiation necrosis, but without producing a discernible increase in tissue superoxide disumtase (SOD) activity. In vitro, MPA pretreatment protected against radiation-induced apoptosis in primary neuronal cultures and increased clonogenic survival of irradiated rat glioma C6 cells, but had no discernible effect on radiation-induced DNA double-strand breaks. MPA, a low molecular weight SOD mimic, significantly increased mitochondrial SOD activity in C6 cells, but not total cellular SOD activity. MPA up-regulated C6 expression of heme-oxygenase 1 (HO-1), an endogenous radioprotectant, but had no effect on HO-1 levels in human astrocytoma U-251 cells, human prostatic carcinoma LNCaP cells, or primary rat brain microvascular endothelial cells in vitro, nor on brain tissue HO-1 expression levels in vivo. Conclusions: Metalloporphyrin antioxidants merit further exploration as adjunctive radioprotectants for cranial radiotherapy/radiosurgery applications, although the potential for tumour protection must be carefully considered.

Cognitive dysfunction, a significant complication after subarachnoid hemorrhage (SAH), affects up to 60% of survivors. We hypothesized that oral simvastatin would improve vestibulomotor function and reduce cognitive dysfunction after experimental SAH in the rat, and explored the effects of simvastatin on vasospasm and regional cerebral blood flow (rCBF). In total 160 rats were enrolled. Randomization to simvastatin or vehicle occurred after double intracisternal blood injections. Effects of simvastatin 10 mg/kg/d (SV10), simvastatin 1.5 mg/kg/d, or vehicle on rotarod, Morris water maze, neuronal survival, cerebral arterial diameter, and rCBF were determined by a blinded observer (n=15/group). A dose dependent response to simvastatin was observed, with more rapid improvement in vestibulomotor function, less basilar arterial vasospasm, and improved cortical neuronal survival with SV10. However, rotarod performance in the SV10 group deteriorated after 1 week, which correlated with the increased plasma creatine kinase levels (r=-0.737; P=0.0002). Furthermore, when simvastatin was discontinued after 2 weeks, the usual treatment duration in SAH clinical trials, rotarod performance deteriorated acutely, rCBF returned to control values, and no long-term benefit was observed in terms of visual spatial memory. Continuing simvastatin 1.5 mg/kg/d for 5 weeks resulted in sustained improvement in rotarod performance, reduced escape latency (P=0.001), swimming distance (P=0.002), and swimming speed (P=0.03) versus vehicle (n=12/group). Our results indicate that long-term cognitive dysfunction after experimental SAH in the rat can be reduced by simvastatin. However, treatment had to be extended beyond 2 weeks, the traditional risk period for angiographic vasospasm, to improve long-term outcome.

Sustained oxidative stress is a known sequel to focal cerebral ischemia. This study examined the effects of treatment with a single dose or sustained infusion of the redox-modulating MnPorphyrin Mn(III)TDE-2-ImP(5+) on outcome from middle cerebral artery occlusion (MCAO) in the rat. Normothermic rats were subjected to 90 min MCAO followed by 90 min reperfusion and then were treated with a single intracerebroventricular dose of Mn(III)TDE-2-ImP(5+). Neurologic and histologic outcomes were assessed at 1 or 8 weeks post-ischemia. A single dose of Mn(III)TDE-2-ImP(5+) caused a dose-dependent improvement in histologic and neurologic outcome when assessed 1 week post-ischemia. Mn(III)TDE-2-ImP(5+) afforded preservation of brain aconitase activity at 5.5 hrs after reperfusion onset, consistent with its known antioxidant properties. Mn(III)TDE-2-ImP(5+) also attenuated post-ischemic NF-kappaB activation. Evidence for effects on cerebral infarct size and neurologic function had completely dissipated when rats were allowed to survive for 8 weeks post-ischemia. In contrast, a 1-week continuous intracerebroventricular Mn(III)TDE-2-ImP(5+) infusion caused persistent and substantive reduction in both cerebral infarct size and neurologic deficit at 8 weeks post-ischemia. Pharmacologic modulation of post-ischemic oxidative stress is likely to require sustained intervention for enduring efficacy in improving neurologic and histologic outcome from a transient focal ischemic insult.