Intracerebroventricular delivery of self-complementary adeno-associated virus serotype 9 to the adult rat brain.

Gene therapy for the central nervous system is poised to become a powerful treatment for numerous neurological disorders. Adeno-associated viral vectors based on serotype 9 (AAV9) have proven themselves to be strong candidates for delivering gene-based therapies throughout the brain and spinal cord when administered intravenously, intrathecally, intracisternally, and intracerebroventricularly (i.c.v.). Previous studies of i.c.v.-delivered self-complimentary AAV9 have been performed in neonatal mice with delivery of a single dose. However, before clinical trials can be considered, more information is required about the dose-response relationship for transduction efficiency in adult animals. In the current study, three doses of self-complementary AAV9 were administered to adult rats. High levels of transduction were observed in the hippocampus, cerebellum and cerebral cortex, and transduction increased with increasing dosage. Both neurons and astrocytes were transduced. There was no evidence of astrocytosis at the doses tested. Preliminary results from pigs receiving i.c.v. self-complementary AAV9 are also presented. The results of this study will serve to inform dosing studies in large animal models before clinical testing.

Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs.

Adeno-associated viral vector 9 (AAV9) has recently been shown to penetrate the blood-brain barrier via intravascular administration, making it a good candidate for diffuse gene delivery. However, the potential side effects of systemic delivery are unknown. Intrathecal viral vector administration may be more invasive than intravenous injections, but it requires far less vector and it can be performed on an outpatient basis, making it an ideal route of delivery for clinical translation. A total of 12 domestic farm pigs (<20 kg) underwent a single-level lumbar laminectomy with intrathecal catheter placement for AAV9 delivery. Animals were perfused and the tissue was harvested 30 days after treatment. Gene expression was assessed by anti-green fluorescent protein immunohistochemistry. Although a single lumbar injection resulted in gene expression limited to the lumbar segment of the spinal cord, three consecutive boluses via a temporary catheter resulted in diffuse transduction of motor neurons (MNs) throughout the cervical, thoracic and lumbar spinal cords. We now present the first successful robust transduction of MNs in the spinal cord of a large animal via intrathecal gene delivery using a self-complementary AAV9. These promising results can be translated to many MN diseases requiring diffuse gene delivery.

Electrical stimulation and gene-based neuromodulation for control of medically-refractory epilepsy.

The failure of available antiepileptic medications to adequately control seizures in a substantial number of patients underscores the need to develop novel epilepsy therapies. Recent advancements in technology and the success of neuromodulation in treating a variety of neurological disorders have spurred interest in exploring promising therapeutic alternatives, such as electrical stimulation and gene-based synaptic control. A variety of different stimulation approaches to seizure control targeting structures in the central or peripheral nervous system have been investigated. Most studies have been based on uncontrolled observations and empirical stimulation protocols. Today the vagus nerve stimulator is the only FDA approved adjunctive treatment for epilepsy that utilizes electrical stimulation. Other potential strategies including direct stimulation of the epileptogenic cortex and deep brain stimulation of various targets are currently under investigation. Chronically implanted devices for electrical stimulation have a variety of limitations. First, they are susceptible to malfunction and infection. Second, most systems require battery replacement. Finally, electrical stimulation is incapable of manipulating neuronal function in a transmitter specific fashion. Gene delivery to epileptogenic targets or targets implicated in regulating seizure threshold has been investigated as an alternative means of neuromodulation in animal models. In summary, positive preliminary results and the lack of alternative treatment options provide the impetus for further exploration of electrical stimulation and gene-based therapies in pharmacoresistant epilepsy. Various specific targets and approaches to modulating their activity have been investigated in human studies.

Footshock-induced sensitization of electrically elicited startle reflexes.

The acoustic startle reflex can be facilitated by the presentation of a train of footshocks presented in rapid succession (footshock sensitization). Acoustic startle is a short-latency reflex mediated by a neural circuit consisting of the ventral cochlear nucleus (VCN), ventral nucleus of the lateral lemniscus (VLL), the nucleus reticularis pontis caudalis (RPC), and the spinal cord. The present study sought to determine the point along this pathway where footshocks might ultimately alter neural transmission to affect startle response. Rats were implanted bilaterally with stimulating electrodes in either the VCN, VLL, or RPC. Startle could be elicited acoustically with a noise burst or electrically with a single-pulse stimulus to either the VCN, VLL, or RPC before and after a train of ten 0.6-mA, 500-ms shocks presented at a rate of 1 shocks/s. Startle elicited acoustically or electrically in the VCN or VLL was significantly elevated following shocks. In contrast, startle elicited from the RPC showed no sensitization, even though startle elicited acoustically from the same animals during the same test session was facilitated. These data suggest that footshock sensitization ultimately alters transmission in the startle circuit at the RPC.

Associative learning modifies the shortening reflex in the semi-intact leech Hirudo medicinalis: effects of pairing, predictability, and CS preexposure.

Three experiments addressed the importance of the inter-event relationships of contiguity and contingency for associative learning in the semi-intact leech. It was found that both of these relationships are important for the leech to acquire a learned association between a touch (conditional stimulus, CS) and shock (unconditional stimulus, US). The learning can be extinguished if training is followed by explicitly unpaired presentations of the CS and US, which removes the contiguity between the stimuli. Learning is degraded by the introduction of unpredicted USs, as well as by unreinforced presentations of the CS (CS preexposure), both manipulations reduce the contingency between the CS and US. These results suggest that the associative process in both vertebrates and invertebrates share considerable functional similarity in the inter-event relationships important to learning.

Effects of the phosphodiesterase inhibitor rolipram on the acoustic startle response in rats.

Systemic administration of the phosphodiesterase inhibitor rolipram (0.05-10.0 mg/kg, IP) produced a rapid and dose-related increase in the amplitude of the acoustic startle response in rats. The (-) isomer was more potent than the (+) isomer in enhancing startle amplitude. Rolipram increased startle responses that were elicited by brief electrical stimulation of the ventral cochlear nucleus or nucleus reticularis pontis caudalis, two brainstem relay nuclei of the startle neural circuit. A low (5 micrograms) dose of rolipram produced an excitatory effect on startle following spinal (lumbar intrathecal) infusion but not following supraspinal (lateral ventricle) infusion. Rolipram (0.5 mg/kg, IP) excitation of startle was not blocked by drugs which differentially disrupt the release of monoamines (DSP4, reserpine + alpha-methyl-para-tyrosine, reserpine + para-chloro-phenylalanine) or by drugs which differentially block monoamine receptors (haloperidol, prazosin, idazoxan, cinanserin, or cyproheptadine). The marked increase in startle seen following systemic rolipram injection is attributable, at least in part, to an action in the lumbar spinal cord that directly or indirectly facilitates neural transmission along the reticulospinal component of the startle reflex neural pathway. The startle reflex should be a useful behavioral test system for studying the mechanism of action of rolipram and related compounds purported to selectively inhibit calmodulin-independent forms of phosphodiesterase.

Epidural abscess: a delayed complication of esophageal stenting for benign stricture.

A case of anterior cervical epidural abscess associated with perforation of an endoscopically placed esophageal stent is presented. Although delayed esophageal perforation is a known complication of endoscopic stenting, no cases presenting with epidural abscess have yet been reported. The increasing application of endoscopic stenting for benign esophageal strictures provides greater opportunity for this type of delayed complication.

Blockade of the spinal excitatory effect of cAMP on the startle reflex by intrathecal administration of the isoquinoline sulfonamide H-8: comparison to the protein kinase C inhibitor H-7.

Drugs thought to inhibit the actions of protein kinase C (PKC) and cAMP dependent protein kinase (A-kinase) were infused intrathecally into the subarachnoid space of the lumbar region of the spinal cord, and the effects on acoustic startle were measured. Previous work has shown that intrathecal infusion of drugs thought to increase cAMP increase the startle response. The present experiment evaluated whether inhibition of A-kinase would prevent this effect. Rats were infused with the isoquinoline sulfonamide, H-8 (360 nmol) or vehicle (50% dimethyl sulfoxide), 30 min prior to infusion of 100 nmol of dibutyryl cAMP. By itself, H-8 had little effect on startle, but completely blocked the normal excitatory effect of dibutyryl cAMP on startle. In contrast, the isoquinoline sulfonamide, H-7, which is less active in blocking A-kinase, but more active in blocking PKC, did not block dibutyryl cAMP. Moreover, H-8 did not block the excitatory effect of intrathecal infusion of the 5-HT1A receptor agonist, 8-OH-dipropylaminotetraline (8-OH-DPAT). Thus, the blockade of dibutyryl cAMP by H-8 appears somewhat specific and suggests an involvement of A-kinase in the excitatory effects of dibutyryl cAMP on the acoustic startle response. In a second experiment, it was found that administration of the isoquinoline sulfonamide H-7 caused a marked, dose-dependent (150-800 nmol) facilitation of the startle reflex in comparison with its vehicle. Tris buffer (0.1 M). Like H-7, another PKC inhibitor, GT1b (20 nmol) produced a marked increase in the startle reflex versus its vehicle, 0.01 M phosphate buffer.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential blockade of early and late components of acoustic startle following intrathecal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-2-amino-5-phosphonovaleric acid (AP-5).

The present study investigated the individual contributions of spinal cord N-methyl-D-aspartate (NMDA) and non-NMDA receptors to the acoustic startle reflex in rats. The first experiment measured whole body acoustic startle before and after intrathecal infusion of various doses of either the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (AP-5), or the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both compounds depressed startle in a dose-dependent fashion with similar potencies. A second experiment measured startle electromyographically (EMG) in the quadriceps femoris muscle complex in the hindlimbs during auditory stimulation to characterized the effects of these two compounds on the early (approximately 8 ms) or late (approximately 15 ms) EMG components of the startle response. CNQX preferentially blocked the early EMG component of startle, whereas AP-5 preferentially blocked the late component. These results suggest that the acoustic startle reflex involves an early EMG component mediated by spinal non-NMDA receptors, and a late EMG component mediated by spinal NMDA receptors.

Use of factor IX complex in warfarin-related intracranial hemorrhage.

OBJECTIVE: Anticoagulation-treated patients presenting with intracranial hemorrhage, including subdural hematoma, epidural hematoma, subarachnoid hemorrhage, and intracerebral hemorrhage, require urgent correction of their coagulopathy to prevent worsening hemorrhage and to facilitate surgical intervention when necessary. In this study, we compared the use of fresh frozen plasma (FFP) with that of Factor IX complex concentrate (FIXCC) to achieve rapid correction of warfarin anticoagulation. METHODS: Patients admitted to a tertiary care center with computed tomography-proven intracranial hemorrhage and a prothrombin time of more than 17 seconds were considered for inclusion in the study protocol. Complete data sets were obtained for eight patients randomized to treatment with FFP and five patients randomized to treatment with FFP supplemented with FIXCC. The prothrombin time and International Normalized Ratio were measured every 2 hours for 14 hours. Correction of anticoagulation was defined as an International Normalized Ratio of < or =1.3. RESULTS: A difference in repeated International Normalized Ratio measurements during the first 6 hours of correction was observed between the FIXCC and FFP groups (P < 0.03). The rate of correction was greater (P < 0.01) and the time to correction was shorter (P < 0.01) for the FIXCC-treated group. No difference in neurological outcomes was detected between groups, but a higher complication rate was observed for the FFP-treated group. CONCLUSION: The use of FIXCC accelerated correction of warfarin-related anticoagulation in the presence of intracranial hemorrhage.

Characterization of adenoviral gene expression in spinal cord after remote vector delivery.

OBJECTIVES: Recent work has established that the remote injection of attenuated adenoviral vectors may result in central nervous system (CNS) gene expression. These studies suggest that virus passes through peripheral nerves into the CNS. The present experiment attempts to characterize this phenomenon systematically. METHODS: Spinal cord cells staining for the reporter gene beta-galactosidase were histologically quantified after microinjection of the viral vector Ad5RSVntLacZ into rat footpad, muscle, or sciatic nerve. The effects of injection location, titer, and time, as well as nerve crush and dexamethasone, were examined. RESULTS: Sciatic nerve viral vector injection results in significantly higher CNS uptake than intramuscular and subcutaneous injections (P < 0.05). Nerve crush injury caused a time-dependent reduction in spinal cord gene uptake after sciatic nerve adenoviral injection (P < 0.05). Neuronal staining reaches its peak at 6 days after injection (P < 0.002). Peripheral nerve delivery to the CNS increases with augmented titers (P < 0.03). Finally, gene expression is augmented by administration of dexamethasone (P < 0.0001). CONCLUSION: Remote adenoviral vector injection represents a potential method for spinal cord gene therapy that avoids any manipulation of CNS tissue.

Remote adenoviral gene delivery to the spinal cord: contralateral delivery and reinjection.

OBJECTIVE: This study characterizes the distribution of adenoviral genes in the spinal cord after viral vector injection into the sciatic nerve. It also evaluates the ability of repeated adenoviral sciatic nerve injections to prolong gene expression in the spinal cord. METHODS: Rat sciatic nerves were unilaterally coinjected with the retrograde tracer Fluoro-Gold (Fluorochrome, Inc., Denver, CO) and the adenoviral vector Ad5RSVntLacZ. The distribution of adenoviral gene expression in the spinal cord was compared with that of Fluoro-Gold. Next, levels of gene expression in the sciatic nerve and spinal cord were compared after single and repeated injections of Ad5RSVntLacZ. Finally, remote spinal cord gene expression in naive animals was compared with expression in animals that had been pretreated with subcutaneous Ad5RSVntLacZ inoculation. RESULTS: Viral gene expression was detected in all quadrants of the spinal cord gray matter, whereas Fluoro-Gold was detected only in the ipsilateral ventral horn (n = 5). This remote delivery was blocked by sciatic nerve transection (n = 10). Viral gene expression occurred in the sciatic nerve after both initial and repeated injections, whereas remote gene expression in the spinal cord was observed only after primary sciatic nerve injection (n = 24; P < 0.003). As with repeated sciatic nerve injections, subcutaneous inoculation with Ad5RSVntLacZ blocked subsequent remote spinal cord gene delivery (n = 8; P < 0.05). CONCLUSION: Remote viral gene delivery occurs in neurons without direct sciatic nerve projections but is dependent on intact peripheral nerves. Repeated injections fail to boost spinal cord gene expression, because of immune recognition of reinjected virus.

Adenoviral nerve growth factor and beta-galactosidase transfer to spinal cord: a behavioral and histological analysis.

OBJECT: The present study characterizes the time course and loci of gene expression induced by the administration of adenoviral vectors into spinal cord. Although a marked inflammatory response to these vectors occurred, no effect on spinal cord function was seen in the 1st postoperative week. The expression of transgenic genes delivered by viral vectors is being exploited throughout the nervous system. The present study utilized adenoviral vectors containing the Rous sarcoma virus (RSV) promoter and a nuclear localization signal to achieve transgenic expression in mammalian spinal cord. METHODS: Initial experiments utilizing the vector Ad.RSVlacZ (10(12) particles/ml) injected into the region of the central canal resulted in viral gene expression stretching over approximately 1.2 cm of spinal cord. Gene expression was first detected 3 days following viral administration and lasted until postinjection Day 14 with peak expression at Day 7. A variety of cell types in both white and gray matter expressed lacZ. Transgenic expression of the neurotrophin nerve growth factor (NGF) was achieved using injections of Ad.RSVNGF. On histological examination mononuclear inflammatory infiltrate and gliosis were revealed surrounding the injection sites of spinal cords receiving adenovirus but not vehicle. To assess spinal cord function during viral gene expression, animals previously trained in an operant runway task were tested at 7 days postinjection (the peak of viral gene expression) and demonstrated no changes in spinal cord function. CONCLUSIONS: Results of this study using adenoviral neurotrophic gene transfer indicate that it provided an effective tool for the delivery of potentially therapeutic proteins to the injured or diseased spinal cord.

Spinal vs. supraspinal sites of action of the alpha 2-adrenergic agonists clonidine and ST-91 on the acoustic startle reflex.

Previous work has shown that stimulation of alpha 2-adrenergic receptors depresses the startle responses in rats. The present study suggests that this depressant effect involves supraspinal rather than spinal alpha 2-adrenergic receptors because intraventricular but not intrathecal infusion of the hydrophilic alpha 2-adrenergic agonist ST-91 depressed the acoustic startle reflex. To determine the point in the acoustic startle pathway where alpha 2-adrenergic receptor activation might ultimately alter neural transmission, startle responses were elicited electrically from different points along the acoustic startle pathway after systemic administration of clonidine. Clonidine depressed acoustically-elicited startle and startle elicited by electrical stimulation of the ventral cochlear nucleus to a comparable magnitude and over a similar time course. It also partially depressed startle elicited by electrical stimulation of the nucleus reticularis pontis caudalis (RPC). Taken together, these data suggest that alpha 2-adrenergic stimulation depresses startle by acting on supraspinal receptors, but that this effect is ultimately expressed, at least in part, by actions at both spinal and brainstem levels of the acoustic startle response pathway. The results are compared to other drugs known to affect the startle reflex.

Neuro-glial hamartoma of the suprasellar cistern.

A unique case of a suprasellar hamartoma in a 29-year-old woman is presented. The lesion was discovered in the context of a work-up for amenorrhea that had lasted 1 year and was resistant to clomiphene and medroxyprogesterone acetate treatment. Magnetic resonance imaging (MRI) revealed a 1.2-cm anterior suprasellar lesion with no apparent connection to the hypothalamus or hypophysis. She underwent surgical resection of the mass. Pathologic examination revealed randomly arranged mature neurons, glial tissue, and myelinated fibers. There was no evidence of gonadotropin-releasing hormone producing neurons on immunohistochemical studies. Postoperative MRI showed complete resection of the lesion, and 1 year later mensus resumed off medication.

Urinary retention following routine neurosurgical spine procedures.

BACKGROUND: The rate and duration of urinary retention after routine cervical and lumbar spine procedures were studied. METHODS: Preoperative, intraoperative, and postoperative factors related to urinary retention (age, sex, duration of operation, medications, Foley use, hospital stay, and cost) were analyzed in a retrospective review of 503 patients' charts. RESULTS: Urinary retention occurred 38% of the time following cervical and lumbar spine procedures. Advanced age and preoperative beta blockers contributed to a higher incidence of urinary retention. Preoperative anti-inflammatory medications and narcotic analgesics reduced the frequency of urinary retention. The duration of urinary retention was prolonged in older patients and patients who underwent intraoperative Foley catheterization. Urinary retention contributed to longer hospitalization and increased hospital costs. CONCLUSIONS: Transient urinary retention is a common complication of routine neurosurgical spine procedures that prolongs hospital stays and increases the costs of hospitalization.

Anatomically discrete functional effects of adenoviral clostridial light chain gene-based synaptic inhibition in the midbrain.

The gene for the Light Chain fragment of Tetanus Toxin (LC) induces synaptic inhibition by preventing the release of synaptic vesicles. The present experiment applied this approach within the rat midbrain in order to demonstrate that LC gene expression can achieve functionally and anatomically discrete effects within a sensitive brain structure. The deep layers of the superior colliculus/deep mesencephalic nucleus (dSC/DpMe) that are located in the rostral midbrain has been implicated in fear-induced increase of the acoustic startle reflex (fear potentiated startle) but exists in close proximity to neural structures important for a variety of critical functions. The dSC/DpMe of adult rats was injected bilaterally with adenoviral vectors for LC, green fluorescent protein, or vehicle. Synaptobrevin was depleted in brain regions of adenoviral LC expression. LC gene expression in the dSC/DpMe inhibited the increase in startle amplitude seen with the control viral infection, and blocked context-dependent potentiation of startle induced by fear conditioning. Although LC gene expression reduced the absolute amount of cue-specific fear potentiated startle, it did not decrease percent potentiated startle to a cue, nor did it reduce fear-induced contextual freezing, nonspecific locomotor activity, or general health, indicating that its effects were functionally and anatomically specific. Thus, vector-driven LC expression inhibits the function of deep brain nuclei without altering the function of surrounding structures supporting its application to therapeutic neuromodulation.

Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination.

Clostridial neurotoxins have assumed increasing importance in clinical application. The toxin's light chain component (LC) inhibits synaptic transmission by digesting vesicle-docking proteins without directly altering neuronal health. To study the properties of LC gene expression in the nervous system, an adenoviral vector containing the LC of tetanus toxin (AdLC) was constructed. LC expressed in differentiated neuronal PC12 cells was shown to induce time- and concentration-dependent digestion of mouse brain synaptobrevin in vitro as compared to control transgene products. LC gene expression in the rat lumbar spinal cord disrupted hindlimb sensorimotor function in comparison to control vectors as measured by the Basso-Beattie-Bresnahan (BBB) scale (P<0.001) and rotarod assay (P<0.003). Evoked electromyography (EMG) showed increased stimulus threshold and decreased response current amplitude in LC gene-transferred rats. At the peak of functional impairment, neither neuronal TUNEL staining nor reduced motor neuron density could be detected. Spontaneous functional recovery was observed to parallel the cessation of LC gene expression. These results suggest that light chain gene delivery within the nervous system may provide a nondestructive means for focused neural inhibition to treat a variety of disorders related to excessive synaptic activity, and prove useful for the study of neural circuitry.

A behavioral analysis of habituation and sensitization of shortening in the semi-intact leech.

We have previously demonstrated that the shortening reflex of the leech Hirudo medicinalis displays habituation, dishabituation, and sensitization. In this paper we demonstrate that the shortening reflex of the semi-intact animal also displays these phenomena. In the first experiment we found that the magnitude of the touch-elicited shortening reflex decreased as a result of repeated stimulations of the skin every 2 min. The second experiment examined the change in this reflex as a function of the interstimulus interval (ISI). The reflex failed to decrease when the ISI was 10 sec, but decreased significantly when the ISI was either 45 or 360 sec. Finally, in a third experiment we found that the presentation of noxious stimuli prior to habituation training prevented habituation. Thus, as we have observed previously in the intact animal, the semi-intact animal displays habituation, dishabituation, and sensitization.

Somatotropinoma infarction during octreotide therapy leading to bilateral cavernous sinus syndrome.

The cyclic somatostatin analog, octreotide, forms the mainstay of medical treatment for acromegaly. In addition to lowering circulating growth hormone levels and shrinking tumor size, octreotide may provide symptomatic relief of headaches associated with growth hormone secreting tumors. The majority of reported complications of octreotide therapy are gastrointestinal and metabolic. The present case illustrates the development of acute bilateral cavernous sinus syndrome with loss of eye movement bilaterally during octreotide therapy. Serial MRI examination suggest tumor infarction as the etiology. The symptoms resolved over 2 months as the tumor shrunk in size and growth hormone was dramatically reduced.