MR-guided parenchymal delivery of adeno-associated viral vector serotype 5 in non-human primate brain.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with AAV5 viral vector after parenchymal delivery. AAV5-CAG-GFP (1 × 1013 vector genomes per milliliter (vg ml-1)) was bilaterally infused either into putamen, thalamus or with the combination left putamen and right thalamus. Robust expression of GFP was seen throughout infusion sites and also in other distal nuclei. Interestingly, thalamic infusion of AAV5 resulted in the transduction of the entire corticospinal axis, indicating transport of AAV5 over long distances. Regardless of site of injection, AAV5 transduced both neurons and astrocytes equally. Our data demonstrate that AAV5 is a very powerful vector for the central nervous system and has potential for treatment of a wide range of neurological pathologies with cortical, subcortical and/or spinal cord affection.

Slow AAV2 clearance from the brain of nonhuman primates and anti-capsid immune response.

Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation.

Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain.

We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.

Magnetic resonance imaging-guided delivery of adeno-associated virus type 2 to the primate brain for the treatment of lysosomal storage disorders.

Gene replacement therapy for the neurological deficits caused by lysosomal storage disorders, such as in Niemann-Pick disease type A, will require widespread expression of efficacious levels of acid sphingomyelinase (ASM) in the infant human brain. At present there is no treatment available for this devastating pediatric condition. This is partly because of inherent constraints associated with the efficient delivery of therapeutic agents into the CNS of higher order models. In this study we used an adeno-associated virus type 2 (AAV2) vector encoding human acid sphingomyelinase tagged with a viral hemagglutinin epitope (AAV2-hASM-HA) to transduce highly interconnected CNS regions such as the brainstem and thalamus. On the basis of our data showing global cortical expression of a secreted reporter after thalamic delivery in nonhuman primates (NHPs), we set out to investigate whether such widespread expression could be enhanced after brainstem infusion. To maximize delivery of the therapeutic transgene throughout the CNS, we combined a single brainstem infusion with bilateral thalamic infusions in naive NHPs. We found that enzymatic augmentation in brainstem, thalamic, cortical, as well subcortical areas provided convincing evidence that much of the large NHP brain can be transduced with as few as three injection sites.

Participation of oxygen and role of exogenous and endogenous sensitizers in the photoinactivation of Escherichia coli by photosynthetically active radiation, UV-A and UV-B.

We studied the mechanisms by which photosynthetically active radiation (PAR) and ultraviolet (UV-A and UV-B) radiation damage Escherichia coli suspended in water. The roles played by oxygen and exogenous and endogenous sensitizers were analyzed by monitoring changes in the physiological state of irradiated cells. Impairment of the cellular functions was more severe in the case of UV radiations. Radiation caused cellular damage in the absence of oxygen. PAR, UV-A, and UV-B radiation induced photobiological and photodynamic reactions mediated by endogenous sensitizers, which significantly shortened the T90 (time needed to reduce a cellular parameter by 90%) based on the growth ability of the cells. In addition, when exogenous sensitizers were present, the photodynamic reactions also had a negative effect on the operation of the electron transport chains. The presence of oxygen might enhance photoinactivation, affecting both the growth ability and the electron transport chains. Endogenous sensitizers were responsible for the noxious action of oxygen. The presence of dissolved organic material played a protective role against the oxygen by absorbing the incident radiation, thereby reducing the energy that reached the endogenous sensitizers.

Tremor is associated with PET measures of nigrostriatal dopamine function in MPTP-lesioned monkeys.

Unilateral intracarotid artery (ICA) MPTP infusion, along with sequential systemic doses of MPTP, produces near complete degeneration of the nigrostriatal pathway on the side of infusion (ipsilateral) and variable levels of damage in the contralateral hemisphere accompanied by varying levels of parkinsonism (overlesioned hemiparkinsonian model). Positron emission tomography and the dopamine (DA) metabolism tracer [(18)F]6-fluoro-l-m-tyrosine (FMT) were used to evaluate the relationship between DA metabolism and clinical features of parkinsonism in 14 overlesioned hemiparkinsonian monkeys. Monkeys were rated on a parkinsonian scale that included ratings of bradykinesia, fine motor skills (FMS), and rest tremor. Because the monkeys tended to show more severe clinical signs on the side of the body contralateral to ICA MPTP infusion, we calculated asymmetry scores for each of the clinical features as well as for FMT uptake (K(i)) in the caudate and putamen. Tremor asymmetry was associated with FMT uptake asymmetry in the putamen. No such relationship was observed for FMS or bradykinesia. The overall severity of tremor (mild, moderate/severe) was associated with FMT uptake in the caudate and putamen. Postmortem biochemical analysis for a subset of monkeys showed that the monkeys with moderate/severe tremor had significantly lower DA levels in both caudate and putamen than those with mild tremor. In addition, K(i) values were significantly correlated with DA levels in both caudate and putamen. These findings support the idea that nigrostriatal degeneration contributes to rest tremor.

Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach.

Using an approach that combines gene therapy with aromatic l-amino acid decarboxylase (AADC) gene and a pro-drug (l-dopa), dopamine, the neurotransmitter involved in Parkinson's disease, can be synthesized and regulated. Striatal neurons infected with the AADC gene by an adeno-associated viral vector can convert peripheral l-dopa to dopamine and may therefore provide a buffer for unmetabolized l-dopa. This approach to treating Parkinson's disease may reduce the need for l-dopa/carbidopa, thus providing a better clinical response with fewer side effects. In addition, the imbalance in dopamine production between the nigrostriatal and mesolimbic dopaminergic systems can be corrected by using AADC gene delivery to the striatum. We have also demonstrated that a fundamental obstacle in the gene therapy approach to the central nervous system, i.e., the ability to deliver viral vectors in sufficient quantities to the whole brain, can be overcome by using convection-enhanced delivery. Finally, this study demonstrates that positron emission tomography and the AADC tracer, 6-[(18)F]fluoro-l-m-tyrosine, can be used to monitor gene therapy in vivo. Our therapeutic approach has the potential to restore dopamine production, even late in the disease process, at levels that can be maintained during continued nigrostriatal degeneration.

Technique for bilateral intracranial implantation of cells in monkeys using an automated delivery system.

Intracerebral grafting combined with gene transfer may provide a powerful technique for local delivery of therapeutic agents into the CNS. The present study was undertaken to: (i) develop a reliable and reproducible automated cell implantation system, (ii) determine optimal implantation parameters of cells into the striatum, (iii) determine upper safe limits of cellular implantation into the neostriatum of monkeys. Autologous fibroblasts were infused into six sites of the striatum in nonhuman primates (Macaca mulatta, n = 11). Twenty-six-gauge cannulae were inserted vertically through cortical entry sites into the striatum (two sites in the caudate nucleus and four sites in the putamen) at predefined coordinates based on magnetic resonance imaging (MRI). The cannulae were guided by an electronically operated, hydraulic micropositioner and withdrawn at controlled rates, while cells (5, 10, 20, 40, or 80 microl/site) were infused simultaneously. Varying infusion rates and cell concentrations were also evaluated. Visualization and evaluation of graft placement were performed using contrast MRI at 3-5 days postsurgery. Animals were monitored for signs of clinical complications and sacrificed 2 weeks following surgery. Postimplantation MRI revealed a tissue mass effect of the implant with shifting of midline, edema, and infiltration of the white tracts at 40 and 80 microl/site. In addition, these animals developed transient hemiparesis contralateral to the implant site. MRI of animals grafted with 20 microl/site exhibited columnar-shaped implants and evidence of infiltration into white matter tracts possibly due to a volume effect. No clinical side effects were seen in this group. At 14 days postsurgery, MRI scans showed consistent columnar grafts (measuring approximately 5 mm in height) throughout the striatum in animals implanted with 5 or 10 microl/site. No signs of clinical side effects were associated with these volumes and postmortem histological examination confirmed MRI observations. Optimal surgical parameters for delivery of cells into the striatum consist of a graft volume of 10 microl/site, an infusion rate of 1.6 microl/min, a cell concentration of 2.0 x 10(5) cells/microl, and a cannula withdrawal rate of 0.75 mm/min. These results show that infusion of cells into the striatum can be done in a safe and routine manner.

Humic Materials Offer Photoprotective Effect to Escherichia coli Exposed to Damaging Luminous Radiation.

The behavior of Escherichia coli immersed in aqueous systems amended with humic acids, under PAR, UV-A, UV-B, and simulated solar radiation was examined. Culturability, ability to elongate, functioning of the electron transport systems, and glucose uptake were assessed. Humic substances in the range from 1 to 50 mg L-1 protected cells from photoinactivation. Decrease in culturability and cellular activities was significantly (p <0.05) less in the presence of humic material. However, humic acids were not used as nutrients. Neither irradiated nor nonirradiated humic solutions (50 mg L-1) supported the growth of 105 cells ml-1. However, humic acids dissolved in 0.9% NaC1 efficiently absorbed light over wavelengths from 270 to 500 nm. Also, a photoprotective effect against simulated sunlight was observed when humic acids were not in contact with but rather enveloped the cellular suspensions in double-wall microcosms. The protection afforded by humic acids against luminous radiation likely derives from their ability to absorb these radiations and hence reduces the amount of energy reaching the cells.

Dopamine transporter loss and clinical changes in MPTP-lesioned primates.

Single photon emission computed tomography (SPECT) and the dopamine (DA) transporter tracer, 2 beta-carboxymethoxy-3 beta-(4-iodophenyl)tropane ([123I]beta-CIT), were used to determine DA transporter density in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-lesioned monkeys with varying degrees of parkinsonism. The clinical stage of parkinsonism corresponded to SPECT measures of striatal DA transporter density suggesting that more severe parkinsonism was associated with a greater degree of dopaminergic terminal degeneration. These findings are similar to those reported earlier using positron emission tomography (PET) and the DA metabolism tracer, 6-[18F]fluoro-L-m-tyrosine (FMT), indicating that both are good methods for evaluating nigrostriatal degeneration in MPTP primate models.

Discharge of disinfected wastewater in recipient aquatic systems: fate of allochthonous bacterial and autochthonous protozoa populations.

The discharge of disinfected effluents affects the bacterivorous ability of protozoa and the effect depends on the disinfectant applied. Chlorine provokes a decrease in the number of protozoa and a delay in the bacterivorous ability. The discharge of ozonated and peracetic acid-treated wastewater provokes only an initial slight decrease in bacterivorous ability. No correlation was found between toxicity values detected using the Microtox assay and the effect of disinfected effluents on freshwater protozoa population. After the disinfection processes, recipient systems (fresh and marine water) have different effects on the survival of Escherichia coli populations discharged to them. The effect of the freshwater recipient system is less negative than the effect provoked by sea-water, and the differences detected depend on the disinfection treatment applied. The wastewater bacterial population as a whole is able to grow after discharge of disinfected wastewater to receiving waters. However, in the absence of predation or competition, the recipient systems exert selection, with rod-shaped bacteria predominating.

A novel MPTP primate model of Parkinson's disease: neurochemical and clinical changes.

Positron emission tomography (PET) and the dopamine (DA) metabolism tracer, [18F]6-fluoro-L-m-tyrosine (FMT) were used to evaluate the relationship between DA metabolism and the clinical stage of parkinsonism monkeys following either unilateral ICA MPTP infusion or unilateral ICA MPTP infusion and subsequent varying sequential systemic doses of MPTP. Clinical stage corresponded to PET measures of striatal DA metabolism, showing the usefulness of the overlesioned hemiparkinsonian monkey as a stable model of various stages of Parkinson's disease (PD).

Increased cardiac fatty acid uptake with dobutamine infusion in swine is accompanied by a decrease in malonyl CoA levels.

OBJECTIVE: Malonyl CoA is an important regulator of fatty acid oxidation in the heart secondary to its ability to inhibit carnitine palmitoyltransferase 1 (CPT 1). Malonyl CoA is produced from acetyl CoA in a reaction catalyzed by acetyl CoA carboxylase (ACC). In this study we determined if alterations in malonyl CoA regulation of fatty acid metabolism are involved in the increase in energy transduction seen following an increase in cardiac work. METHODS: Anesthetized, open-chest, domestic swine were subjected to a 30 min control period followed by a 30 min treatment period with either dobutamine (15 micrograms.kg-1. min-1 i.v.) (n = 6) or saline (n = 6). RESULTS: Heart rate, left ventricular peak dp/dt, and MVO2, were significantly increased in the dobutamine group compared to the saline group during the treatment period. Free fatty acid and glucose uptake were increased 210 and 248%, respectively, in the dobutamine group during the treatment period. Malonyl CoA content was decreased by 55% (from 0.40 +/- 0.05 to 0.18 +/- 0.12 nmol/g wet wt; P < 0.05) with dobutamine treatment, but was not affected by saline treatment. ACC activity was not significantly different between groups (0.31 +/- 0.02 vs. 0.30 +/- 0.04 nmol. min-1. mg protein-1, respectively). The activity of AMP-dependent protein kinase (AMPK), which phosphorylates and inactivates ACC, was also not significantly different in the dobutamine hearts compared to the saline hearts (322 +/- 26 vs. 338 +/- 39 pmol. min-1. mg protein-1, respectively). CONCLUSION: The increased cardiac work following dobutamine infusion is accompanied by a decrease in malonyl CoA levels and an increase in fatty acid uptake. However, the decrease in malonyl CoA cannot be explained by a decrease in ACC activity.

Pyruvate dehydrogenase activity and malonyl CoA levels in normal and ischemic swine myocardium: effects of dichloroacetate.

The purposes of this study were to: (1) assess myocardial pyruvate dehydrogenase (PDH) activity and substrate exchange under well-perfused and ischemic conditions; (2) determine the metabolic effects of an intra-coronary infusion of the PDH activator, dichloroacetate (DCA); and (3) measure the effects of ischemia and DCA on malonyl CoA levels. Experiments were performed in anesthetised open-chest swine under non-ischemic conditions, followed by 40 min with a 60% reduction in left anterior descending coronary artery (LAD) blood flow. Myocardial needle biopsies for measurement of PDH activity were taken after an intracoronary infusion of either saline or DCA (1 mM in LAD blood) under aerobic conditions, and after 37 min of ischemia. Pyruvate dehydrogenase activity was measured with and without maximal activation by swine PDH phosphatase. Malonyl CoA and acetyl CoA were measured after 40 min of LAD ischemia in myocardium from the ischemic DCA- or saline-treated LAD bed, and the non-ischemic untreated left circumflex coronary artery (CFX) perfusion bed. Net glucose, lactate and free fatty acid (FFA) uptakes were measured across the LAD perfusion bed throughout the study. Dichloroacetate treatment increased the amount of active dephosphorylated PDH to 88% of the total activity under aerobic conditions, compared to 55% with saline (P < 0.01). Ischemia did not significantly change PDH activation state in either group. Acetyl CoA and malonyl CoA contents were significantly elevated in ischemic DCA-treated myocardium compared to saline-treated ischemic myocardium. Dichloroacetate treatment significantly lowered rates of myocardial FFA uptake under both aerobic and ischemic conditions, but did not effect glucose uptake or lactate exchange. Free fatty acid uptake was negatively correlated to malonyl CoA levels (r = -0.68) during ischemia. It is proposed that the inhibition of FFA uptake observed with DCA in ischemic myocardium is due to malonyl CoA inhibition of carnitine palmitoyl transferase I.

Cerebrospinal fluid pH and PCO2 rapidly follow arterial blood pH and PCO2 with changes in ventilation.

Changes in ventilatory rate affect arterial blood pH and PCO2 within seconds to minutes, but the corresponding acute changes for cerebrospinal fluid (CSF) pH and PCO2 have been as well documented. Using our previously-described swine model of brain retraction ischemia, we examined changes in arterial and CSF pH and PCO2 with acute changes in ventilation in four animals. Newly developed fluorescent dye technology permitted near-instantaneous recording of CSF pH and PCO2 during acute hyperventilation (end-tidal PCO2 of 20 mm Hg) and acute hypoventilation (end-tidal PCO2 of 50 mm Hg). The Puritan-Bennett 3300 Intra-Arterial Blood Gas Monitor (PB3300) was used with the sensor placed in the CSF in the interhemispheric fissure posterior to the corpus callosum. The following data were gathered at 5, 15, 30, and 60 minutes after the ventilatory change: arterial pH and PCO2, end-tidal CO2, laser-Doppler cerebral blood flow, and CSF pH and PCO2. The baseline (normoventilation) values for arterial and CSF pH and PCO2 in swine were comparable to those in humans: arterial pH 7.44 and PCO2 43 mm Hg; CSF pH 7.31 and PCO2 55 mm Hg. Changes in pH and PCO2 with hyperventilation and hypoventilation occurred rapidly in both arterial blood and CSF. Steady-state values were reached within 15 minutes for hypoventilation, and 30 minutes for hyperventilation. The correlation between arterial and CSF values for both pH and PCO2 at 5, 15, 30, and 60 minutes were all very highly significant (P < 0.001) except for arterial and CSF PCO2 at 5 minutes (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

A review of brain retraction and recommendations for minimizing intraoperative brain injury.

Brain retraction is required for adequate exposure during many intracranial procedures. The incidence of contusion or infarction from overzealous brain retraction is probably 10% in cranial base procedures and 5% in intracranial aneurysm procedures. The literature on brain retraction injury is reviewed, with particular attention to the use of intermittent retraction. Intraoperative monitoring techniques--brain electrical activity, cerebral blood flow, and brain retraction pressure--are evaluated. Various intraoperative interventions--anesthetic agents, positioning, cerebrospinal fluid drainage, operative approaches involving bone resection or osteotomy, hyperventilation, induced hypotension, induced hypertension, mannitol, and nimodipine--are assessed with regard to their effects on brain retraction. Because brain retraction injury, like other forms of focal cerebral ischemia, is multifactorial in its origins, a multifaceted approach probably will be most advantageous in minimizing retraction injury. Recommendations for operative management of cases involving significant brain retraction are made. These recommendations optimize the following goals: anesthesia and metabolic depression, improvement in cerebral blood flow and calcium channel blockade, intraoperative monitoring, and operative exposure and retraction efficacy. Through a combination of judicious retraction, appropriate anesthetic and pharmacological management, and aggressive intraoperative monitoring, brain retraction should become a much less common source of morbidity in the future.

Corpus callosotomy effects on cerebral blood flow and evoked potentials (transcallosal diaschisis).

The role of the corpus callosum in diaschisis was examined through the acute effects of stereotactic corpus callosum section on cerebral blood flow and somatosensory or auditory evoked potentials bilaterally during unilateral brain retraction ischemia, using a previously reported swine model. Cerebral blood flow and evoked potential amplitude contralateral to retraction increased during retraction with the corpus callosum intact, compared with post-callosal section values. With retraction following callosal section, there was no increase in cerebral blood flow or evoked potential amplitude contralateral to retraction. Diaschisis during the early stages of a focal, unilateral injury takes the form of a contralateral disinhibition (as measured by cerebral blood flow and evoked potentials), an effect which is lost following callosal section.

Effects of mannitol on cerebral blood flow, blood pressure, blood viscosity, hematocrit, sodium, and potassium.

In our miniature swine model of brain retraction ischemia under conditions simulating the neurosurgical operating room, we studied the effects of bolus mannitol (2 g/kg) administration on cerebral blood flow, blood pressure, blood viscosity, hematocrit, sodium, and potassium serially for 4 hours following administration, at which time a second bolus was administered. Both viscosity and hematocrit were significantly decreased transiently following both the first and second boluses. Sodium was decreased for 30 minutes following the first bolus, 15 minutes following the second bolus, and increased at 150 minutes and later following the second bolus. There was a mild decrease in blood pressure and a mild increase in cerebral blood flow following mannitol, but little difference between the first hour following a bolus (when the viscosity and hematocrit were decreased) and hours 2-4 (when they were near baseline). Mannitol's effects on blood pressure and cerebral blood flow probably depend on factors in addition to its effects on blood viscosity and hematocrit. The results are discussed in light of previous findings that bolus mannitol administration may improve cerebral blood flow in ischemia, but does not appear to benefit the preservation of brain electrical activity.