Continuous intrathecal fluid infusions elevate nerve growth factor levels and prevent functional deficits after spinal cord ischemia

Abstract

Continuous intracerebroventricular or intrathecal infusions of neurotrophic factors have been reported to prevent neuronal degeneration, stimulate axonal sprouting and ameliorate behavioral deficits in various models of CNS injury and aging. In the present study, the ability of intrathecal infusions of recombinant human nerve growth factor (NGF) to reduce functional deficits following spinal cord ischemia was investigated. Adult rabbits underwent intrathecal cannulation and continuous infusions of either 300 μg/ml recombinant human NGF or artificial CSF (vehicle) at a rate of 143 μl/day for 7 days prior to induction of spinal cord ischemia. Continuous infusions were maintained after induction of ischemia. Four days later, both NGF-treated and vehicle-infused subjects showed a significant amelioration of functional motor deficits compared to lesioned, non-infused subjects (P<0.05). The average duration of tolerated ischemia increased from 23.4±1.8 min in lesioned, non-infused subjects to 35.5±3.1 min in lesioned, artificial CSF-infused subjects and 35.6±4.7 min in NGF-infused subjects (mean±S.E.M.). Significantly elevated NGF protein levels were attained within the spinal cords of both NGF-treated subjects and artificial CSF-infused subjects, although levels were substantially higher in NGF-treated subjects (9.8±3.8 ng/g in NGF-infused vs. 2.0±0.4 ng/g in vehicle-infused and only 0.4±0.2 ng/g in lesioned, non-infused animals). These findings indicate that the process of intrathecal cannulation and fluid infusion elicits alterations in the spinal cord environment that are neuroprotective, including spontaneous elevations in NGF levels.

Introduction

Neurotrophic factors prevent neuronal degeneration and promote axonal growth in responsive cell populations in various regions of the developing and adult mammalian nervous system [7]. Neurotrophic factors such as nerve growth factor (NGF) help to maintain normal neuronal function and also sustain the phenotype of neurons following injury to the nervous system. Neurotrophin levels become elevated in some regions of the nervous system after trauma, as a result of either increased production or diminished utilization by responsive neurons [22]. For example, Schwann cells in injured peripheral nerves upregulate expression of nerve growth factor (NGF), brain-derived neurotrphic factor (BDNF) and ciliary neurotrophic factor (CNTF) mRNA and increase NGF protein production [18]. NGF levels rise in the hippocampus following axotomy of cholinergic inputs, presumably as a result of reduced uptake and retrograde transport by cholinergic terminal [15]. NGF administration prevents degeneration of septal cholinergic neurons after axotomy [13], and implantation into the brain of fibroblasts that are genetically modified to secrete NGF reduces histopathology following axonal transection [29] or excitotoxic injury [8]. NGF infusion has also been shown to attenuate cognitive impairments but not motor impairments in rats after traumatic brain injury [26]. NGF levels are elevated following cerebral ischemia in the hippocampal CA1 region and decreased in other brain regions [12], and administration of exogenous NGF can reduce neuronal necrosis following cerebral ischemia [25], [27], [34]. It therefore appears that the production of neurotrophic factors is a feature of several types of CNS injury, and that neuronal injury may be attenuated by this response.

Neurotrophins have often been delivered to the CNS by cannulating the ventricular system [13], [16], [25], [27] or intrathecal space [11], [31], or by intraparenchymal infusion [26]. Although NGF appears to ameliorate the consequences of neuronal injury, the infusion process itself may damage the CNS or elicit inflammatory processes in the CNS, potentially resulting in upregulated production of substances such as neurotrophic factors [6], [19], [20]. Neuron survival after injury has been improved by infusion of artificial cerebrospinal fluid alone [10], [25], [27], as well as by implantation of atelocollagen pellets directly into the hippocampus [34]. This protective effect is analogous to findings that the placement of sham grafts into the striatum can induce behavioral recovery after MPTP-induced dopamine neuron lesions in primates, mimicking the beneficial effects of fetal tissue grafts [6], [20]. It has been speculated that a mechanism of recovery following tissue grafting to the CNS may be elicitation of neurotrophin production in the host brain resulting from limited damage of the grafting procedure itself [6], [20].

In the present study, we sought to determine whether central infusions of nerve growth factor (NGF) or artificial cerebrospinal fluid (aCSF) would ameliorate functional deficits in a well-characterized model of adult rabbit spinal cord ischemia [3], [35]. Although NGF appears to limit hippocampal ischemic injury, the hippocampus is more sensitive to ischemic damage than any other brain regions and is therefore unrepresentative of broader neuronal populations. Thus, it is not clear whether NGF protection also extends to other CNS cell groups. Various neuronal populations exhibit specificity for different neurotrophic factors: for example, basal forebrain cholinergic neurons are primarily responsive to NGF, dopaminergic neurons to glial cell-line derived neurotrophic factor (GDNF), and motor neurons to CNTF, BDNF or GDNF (see e.g. Refs. [10], [17], [23], [24], [30], [31], [32], [33]). In the spinal cord, low-affinity neurotrophin receptors have been detected on injured but not intact motoneurons of the spinal cord [14], suggesting that motor neuronal responses to injury may be regulated by growth factors. Further, sensory projections to the spinal cord express both low- and high-affinity neurotrophin receptors throughout life, and robustly extend new axons when provided with NGF after injury [28]. Finally, reactive astrocytes and microglia express neurotrophins after injury in the CNS, suggesting that more diverse and widespread effects of neurotrophins may act to influence spinal cord responses to injury [1], [5], [21].

To date, few studies that have examined the effects of NGF administration in models of CNS ischemia have also evaluated functional outcomes, despite several reports of NGF-induced neuroprotection. In the present experiment, we evaluated functional neurological outcome 18 h and 4 days after reversible spinal ischemia in rabbits. The intrathecal space was cannulated and infused with NGF or aCSF continuously for 3 days prior to induction of ischemia, and continuously for 4 days thereafter. Functional outcomes and NGF levels in the spinal cords were then assessed.