Piyush M. Patel

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

Inhibition of p75 Neurotrophin Receptor Attenuates Isoflurane-mediated Neuronal Apoptosis in the Neonatal Central Nervous System

Background:Exposure to anesthetics during synaptogenesis results in apoptosis and subsequent cognitive dysfunction in adulthood. Probrain-derived neurotrophic factor (proBDNF) is involved in synaptogenesis and can induce neuronal apoptosis via p75 neurotrophic receptors (p75NTR). proBDNF is cleaved into mature BDNF (mBDNF) by plasmin, a protease converted from plasminogen by tissue plasminogen activator (tPA) that is released with neuronal activity; mBDNF supports survival and stabilizes synapses through tropomyosin receptor kinase B. The authors hypothesized that anesthetics suppress tPA release from neurons, enhance p75NTR signaling, and reduce synapses, resulting in apoptosis. Methods:Primary neurons (DIV5) and postnatal day 5-7 (PND5-7) mice were exposed to isoflurane (1.4%, 4 h) in 5% CO2, 95% air. Apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Dendritic spine changes were evaluated with the neuronal spine marker, drebrin. Changes in synapses in PND5-7 mouse hippocampi were assessed by electron microscopy. Primary neurons were exposed to tPA, plasmin, or pharmacologic inhibitors of p75NTR (Fc-p75NTR or TAT-Pep5) 15 min before isoflurane. TAT-Pep5 was administered by intraperitoneal injection to PND5-7 mice 15 min before isoflurane. Results:Exposure of neurons in vitro (DIV5) to isoflurane decreased tPA in the culture medium, reduced drebrin expression (marker of dendritic filopodial spines), and enhanced Cl-Csp3. tPA, plasmin, or TAT-Pep5 stabilized dendritic filopodial spines and decreased Cl-Csp3 in neurons. TAT-Pep5 blocked isoflurane-mediated increase in Cl-Csp3 and reduced synapses in PND5-7 mouse hippocampi. Conclusion:tPA, plasmin, or p75NTR inhibition blocked isoflurane-mediated reduction in dendritic filopodial spines and neuronal apoptosis in vitro. Isoflurane reduced synapses and enhanced Cl-Csp3 in the hippocampus of PND5-7 mice, the latter effect being mitigated by p75NTR inhibition in vivo. These data support the hypothesis that isoflurane neurotoxicity in the developing rodent brain is mediated by reduced synaptic tPA release and enhanced proBDNF/p75NTR-mediated apoptosis.

Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia.

Background: Several investigations have shown that volatile anesthetics can reduce ischemic cerebral injury. In these studies, however, neurologic injury was evaluated only after a short recovery period. Recent data suggest that injury caused by ischemia is a dynamic process characterized by continual neuronal loss for a prolonged period. Whether isoflurane-mediated neuroprotection is sustained after a longer recovery period is not known. The current study was conducted to compare the effect of isoflurane on brain injury after short (2-day) and long (14-day) recovery periods in rats subjected to focal ischemia. Metbods: Fasted Wistar-Kyoto rats were anesthetized with isoflurane and randomly allocated to an awake (n = 36) or an isoflurane (n = 34) group. Animals in both groups were subjected to focal ischemia by filament occlusion of the middle cerebral artery. Pericranial temperature was servocontrolled at 37°C throughout the experiment. In the awake group, isoflurane was discontinued and the animals were allowed to awaken. In the isoflurane group, isoflurane anesthesia was maintained at 1.5 times the minimum alveolar concentration. After 70 min of focal ischemia, the filament was removed. Animals were killed 2 days (awake, n = 18; isoflurane, n = 17) and 14 days (awake, n = 18; isoflurane, n = 17) after ischemia. The volumes of cerebral infarction and selective neuronal necrosis in the animals were determined by image analysis of hematoxylin and eosin-stained coronal brain sections. Results: Cortical and subcortical volumes of infarction were significantly less in the isoflurane 2-day group (26 ± 23 mm 3 and 17 ± 6 mm 3 , respectively) than in the awake 2-day group (58 ± 35 mm 3 , P < 0.01; and 28 ± 12 mm 3 , P < 0.01, respectively). By contrast, cortical and subcortical volumes of infarction in the awake (41 ± 31 mm 3 and 28 ± 16 mm 3 , respectively) and isoflurane (41 ± 35 mm 3 and 19 ± 8 mm 3 , respectively) 14-day groups were not different (cortex, P = 0.99; subcortex, P = 0.08). The volume of cortical tissue in which selective neuronal necrosis was observed, however, was significantly less in the isoflurane 14-day group (5 ± 4 mm 3 ) than in the awake 14-day group (17 ± 9 mm 3 , P < 0.01). The total number of necrotic neurons in the region of selective neuronal necrosis was significantly smaller in the isoflurane 14-day group than in the awake 14-day group (P < 0.01). Conclusion: Compared with the awake state, isoflurane reduced the extent of infarction assessed 2 days after focal ischemia in rats. At 14 days, however, only selective neuronal necrosis, but not infarction, was reduced by isoflurane. These results suggest that isoflurane delays but does not prevent cerebral infarction caused by focal ischemia. Isoflurane may attenuate the delayed development of selective neuronal necrosis in periinfarct areas in this animal model.

EFFECTS OF PROPOFOL, ETOMIDATE, MIDAZOLAM AND FENTANYL ON MOTOR EVOKED RESPONSES TO TRANSCRANIAL ELECTRICAL OR MAGNETIC STIMULATION IN HUMANS

The effects of propofol, etomidate, midazolam, and fentanyl on motor evoked responses to transcranial stimulation (tc-MERs) were studied in five healthy human volunteers. Each subject, in four separate sessions, received intravenous bolus doses of propofol 2 mg.kg-1, etomidate 0.3 mg.kg-1, midazolam 0.05 mg.kg-1, and fentanyl 3 micrograms.kg-1. Electrical tc-MERs (tce-MERs) were elicited with anodal stimuli of 500-700 V. Magnetic tc-MERs (tcmag-MERs) were elicited using a Cadwell MES-10 magnetic stimulator at maximum output. Compound muscle action potentials were recorded from the tibialis anterior muscle. Duplicate tce-MERs and tcmag-MERs were recorded before and up to 30 min after drug injection. Reproducible baseline tce-MERs (amplitude 4.7 +/- 0.43 (SEM) mV, latency 29.4 +/- 0.35 ms) and tcmag-MERs (amplitude 3.7 +/- 0.43 mV, latency 31.1 +/- 0.39 ms) were obtained in all subjects. Pronounced depression of tce-MER amplitude to 2% of baseline values (P less than 0.01) was observed 2 min after injection of propofol. Thirty minutes after injection of propofol, amplitude depression to 44% of baseline (P less than 0.05) was still present, despite an apparent lack of sedation. Midazolam caused significant (P less than 0.01) amplitude depression, e.g., tcmag-MER to 16% of baseline values 5 min after injection. Significant depression persisted throughout the 30-min study period. Fentanyl did not cause any statistically significant amplitude changes in this small population. Etomidate caused significant but transient depression of tc-MER amplitude. However, there was considerable intersubject variability. Latency did not change significantly after any drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroprotective effects of anesthetic agents

Ischemic neuronal injury is characterized by early death mediated by excitotoxicity and by delayed death caused by apoptosis. Current evidence indicates that volatile agents, barbiturates, and propofol can protect neurons against ischemic injury caused by excitotoxicity. In the case of volatile agents and propofol, neuroprotection may be sustained if the ischemic insult is relatively mild; however, with moderate to severe insults, this neuronal protection is not sustained after a prolonged recovery period. This suggests that volatile agents and propofol do not reduce delayed neuronal death caused by apoptosis. The long-term effects of barbiturates on ischemic cerebral injury are not yet defined. Cerebral ischemia is characterized by continued neuronal loss for a long time after the initial ischemic insult. Therefore, in investigations of cerebral ischemia, the duration of the recovery period should be taken into consideration in the analysis of the neuroprotective effects of anesthetic agents. A combination of different approaches that target specific stages of the evolution of ischemic injury may be required for sustained neuroprotection.

Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1

Caveolae, small invaginations in the plasma membrane, contain caveolins (Cav) that scaffold signaling molecules including the tyrosine kinase Src. We tested the hypothesis that cardiac protection involves a caveolin‐dependent mechanism. We used in vitro and in vivo models of ischemia‐reperfusion injury, electron microscopy (EM), transgenic mice, and biochemical assays to address this hypothesis. We found that Cav‐1 mRNA and protein were expressed in mouse adult cardiac myocytes (ACM). The volatile anesthetic, isoflurane, protected ACM from hypoxia‐induced cell death and increased sarcolemmal caveolae. Hearts of wild‐type (WT) mice showed rapid phosphorylation of Src and Cav‐1 after isoflurane and ischemic preconditioning. The Src inhibitor PP2 reduced phosphorylation of Src (Y416) and Cav‐1 in the heart and abolished isoflurane‐induced cardiac protection in WT mice. Infarct size (percent area at risk) was reduced by isoflurane in WT (30.5±4 vs. 44.2±3, n=7, P<0.05) but not Cav‐1−/− mice (46.6±5 vs. 41.7±3, n=7). Cav‐1−/−mice exposed to isoflurane showed significant alterations in Src phosphorylation and recruitment of C‐terminal Src kinase, a negative regulator of Src, when compared to WT mice. The results indicate that isoflurane modifies cardiac myocyte sarcolemmal membrane structure and composition and that activation of Src and phosphorylation of Cav‐1 contribute to cardiac protection. Accordingly, therapies targeted to post‐translational modification of Src and Cav‐1 may provide a novel approach for such protection.—Patel, H. H., Tsutsumi, Y. M., Head, B. P., Niesman, I. R., Jennings, M., Horikawa, Y. Huang, D., Moreno, A. L., Patel, P. M., Insel, P. A., Roth, D. M. Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin‐1. FASEB J. 21, 1565–1574 (2007)

Effect of isoflurane on neuronal apoptosis in rats subjected to focal cerebral ischemia.

Although isoflurane can reduce ischemic neuronal injury after short postischemic recovery intervals, this neuroprotective efficacy is not sustained. Neuronal apoptosis can contribute to the gradual increase in infarct size after ischemia. This suggests that isoflurane, although capable of reducing early neuronal death, may not inhibit ischemia-induced apoptosis. We investigated the effects of isoflurane on markers of apoptosis in rats subjected to focal ischemia. Fasted Wistar-Kyoto rats were anesthetized with isoflurane and randomly allocated to awake (n = 40) or isoflurane (n = 40) groups. Animals in both groups were subjected to focal ischemia by filament occlusion of the middle cerebral artery for 70 min. Pericranial temperature was servo-controlled at 37°C ± 0.2°C throughout the experiment. In the awake group, isoflurane was discontinued and the animals were allowed to awaken. In the isoflurane group, isoflurane anesthesia was maintained at 1.5 MAC (minimum alveolar anesthetic concentration). Animals were killed 7 h, 1 day, 4 days, or 7 days after reperfusion (n = 10/group/time point). The area of cerebral infarction was measured by image analysis in a hematoxylin and eosin stained section. In three adjacent sections, apoptotic neurons were identified by TUNEL staining and immunostaining for active caspase-9 and caspase-3. Infarct size was smaller in the isoflurane group than the awake group 7 h, 1 day, and 4 days after reperfusion (P < 0.05). However, this difference was absent 7 days after reperfusion. The number of apoptotic (TUNEL, caspase-3, and caspase-9 positive) cells 1 day after ischemia was significantly more in the awake versus isoflurane group. After a recovery period of 4 or 7 days, the number of apoptotic cells in the isoflurane group was more than in the awake group. After 7 days, the number of caspase-3 and -9 positive neurons was more in the isoflurane group (P < 0.05). The data indicate that isoflurane delays but does not prevent the development of cerebral infarction caused by ischemia. Isoflurane reduced the development of apoptosis early after ischemia but did not prevent it at later stages of postischemic recovery.

Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning

Background— Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte–specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo. Methods and Results— Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte–specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3β phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3β and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion. Conclusions— Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.

Intraoperative monitoring of tibialis anterior muscle motor evoked responses to transcranial electrical stimulation during partial neuromuscular blockade.

We studied the feasibility of recording motor evoked responses to transcranial electrical stimulation (tce-MERs) during partial neuromuscular blockade (NMB). In 11 patients, compound muscle action potentials were recorded from the tibialis anterior muscle in response to transcranial electrical stimulation during various levels of vecuronium-induced NMB. The level of NMB was assessed by accelerometry of the adductor pollicis muscle after train-of-four stimulation of the ulnar nerve. The compound muscle action potential was also recorded from the tibialis anterior muscle after direct stimulation of the peroneal nerve (M-response) as an alternative means of assessing the degree of NMB. In all patients, tce-MERs could be recorded reliably during anesthesia with N2O and a continuous infusion of sufentanil (0.5 micrograms.kg-1.h-1). An intact train-of-four was present in all patients, and the amplitude of the first twitch was recorded and designated as the control value. Before administration of vecuronium, the M-response amplitude was 9.6 +/- 3.6 (mean +/- SD) mV, and the tce-MER amplitude was 1.21 +/- 0.66 mV. Although administration of vecuronium (0.05 mg/kg) resulted in loss of the mechanical adductor pollicis response in 8 of the 11 patients, the M-response and the tce-MER remained recordable. Subsequently, during an infusion of vecuronium, adjusted to maintain one or two mechanical responses to train-of-four stimulation, the average M-response to peroneal nerve stimulation was 5.2 +/- 2.5 mV (53% of the control value), and tce-MER amplitude was 0.59 +/- 0.36 mV (59% of the control value).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Dexmedetomidine on Cerebral Blood Flow Velocity, Cerebral Metabolic Rate, and Carbon Dioxide Response in Normal Humans

Background:Dexmedetomidine reduces cerebral blood flow (CBF) in humans and animals. In animal investigations, cerebral metabolic rate (CMR) was unchanged. Therefore, the authors hypothesized that dexmedetomidine would cause a decrease in the CBF/CMR ratio with even further reduction by superimposed hyperventilation. This reduction might be deleterious in patients with neurologic injuries. Methods:Middle cerebral artery velocity (CBFV) was recorded continuously in six volunteers. CBFV, jugular bulb venous saturation (Sjvo2), CMR equivalent (CMRe), and CBFV/CMRe ratio were determined at six intervals before, during, and after administration of dexmedetomidine: (1) presedation; (2) presedation with hyperventilation; at steady state plasma levels of (3) 0.6 ng/ml and (4) 1.2 ng/ml; (5) 1.2 ng/ml with hyperventilation; and (6) 30 min after discontinuing dexmedetomidine. The slope of the arterial carbon dioxide tension (Paco2)–CBFV relation was determined presedation and at 1.2 ng/ml. Results:CBFV and CMRe decreased in a dose-related manner. The CBFV/CMRe ratio was unchanged. The CBFV response to carbon dioxide decreased from 1.20 ± 0.2 cm·s−1·mm Hg−1 presedation to 0.40 ± 0.15 cm·s−1·mm Hg−1 at 1.2 ng/ml. Sjvo2 was statistically unchanged during hyperventilation at 1.2 ng/ml versus presedation (50 ± 11 vs. 43 ± 5%). Arousal for hyperventilation at 1.2 ng/ml resulted in increased CBFV (30 ± 5 to 38 ± 4) and Bispectral Index (43 ± 10 to 94 ± 3). Conclusions:The predicted decrease in CBFV/CMRe ratio was not observed because of an unanticipated reduction of CMRe and a decrease in the slope of the Paco2–CBFV relation. CBFV and Bispectral Index increases during arousal for hyperventilation at 1.2 ng/ml suggest that CMR–CBF coupling is preserved during dexmedetomidine administration. Further evaluation of dexmedetomidine in patients with neurologic injuries seems justified.