Quantitative assessment of respiratory function following contusion injury of the cervical spinal cord.

In this study, we describe a new method for quantitative assessment of phrenic inspiratory motor activity in two models of cervical spinal cord contusion injury. Anesthetized rats received contusion injury either to the descending bulbospinal respiratory pathway on one side of the spinal cord alone (C2 lateralized contusion) or to both the descending pathway, as well as the phrenic motoneuron pool bilaterally (C4/C5 midline contusion). Following injury, respiratory-associated phrenic nerve motor activity was recorded under standardized and then asphyxic conditions. Phrenic nerve efferent activity was rectified, integrated, and quantitated by determining the mean area under the integrated neurograms. The mean integrated area of the four inspiratory bursts recorded just before turning off the ventilator (to induce asphyxia) was determined and divided by the integrated area under the single largest respiratory burst recorded during asphyxia. This latter value was taken as the maximal inspiratory motor response that the rat was capable of generating during respiratory stress. Thus, a percentage of the maximal inspiratory motor drive was established for breathing in control and injured rats under standardized conditions. The results indicate that noninjured rats use 52 +/- 1.8% of maximal inspiratory motor drive under standardized conditions. In C2-contused rats, the results showed that while the percentage of maximal inspiratory motor drive on the noncontused side was similar to the control (55 +/- 4.1%), it was increased on the contused side (78 +/- 2.6%). In C4/5 lesions, the results indicate that this percentage was increased on both sides (77 +/- 4.4%). The results show the feasibility for performing quantitative evaluation of respiratory dysfunction in an animal model of cervical contusion injury. These findings lend to further development of this model for investigations of neuroplasticity and/or therapeutic interventions directed at ameliorating respiratory compromise following cervical spinal cord trauma.

Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters.

In this study, we have evaluated the capacity of recombinant adeno-associated virus (rAAV) vectors, containing cell type-specific promoters, to transduce neurons in vivo in the normal adult rat spinal cord. The neuron-specific enolase (NSE) promoter and the platelet-derived growth factor (PDGF) B-chain promoter were used to direct expression of a 'humanized' form of the gene for green fluorescent protein (GFP). Neuron-specific rAAVs were injected into the mid-cervical regions of adult rat spinal cords. At 10-14 days, expression was detected in all animals and persisted for up to 15 weeks. Immunocytochemical and morphological profiles of transduced cells were consistently neuronal, and there was no evidence of transgene expression in glial elements. Transduction efficiencies for the NSE and PDGF rAAVs were estimated at 15 and 45 infectious particles per GFP-positive neuron, respectively, in the absence of detectable adenovirus. This study strongly supports a role for rAAV vectors in CNS gene therapy and lays the groundwork for delivery of more functional genes to spinal cord neurons as a possible way to enhance spinal cord repair following injury.

Comparison of the growth and fate of fetal spinal iso- and allografts in the adult rat injured spinal cord.

Most studies investigating early fetal CNS graft-host interactions and host immune responses have been performed using intracerebral transplantation paradigms. The purpose of this study was to establish the early developmental dynamics of fetal graft integration with the injured host spinal cord and to determine whether fetal allografts in this environment are subject to rejection. ACI rat fetal spinal cord (FSC) tissue was grafted into acute lesion cavities of adult WF rat spinal cords. Graft development and/or rejection was followed from 1 to 45 days posttransplantation with morphometric, histological, and immunocytochemical methods. We determined that all FSC grafts in acute resection lesions of the adult rat spinal cord undergo an early substantial cellular attrition, but following favorable attachment to healthy host tissue margins, they rebound and grow to fill the lesion cavity by approximately 45 days. We also determined that FSC allografts into nonimmunosuppressed adult recipients are consistently rejected, but only after an early period of growth and maturation. The onset of rejection is characterized by extensive cellular infiltration coincidental with graft and host MHC antigen expression. The implications of delayed graft development and graft-host integration are discussed relative to interconnectivity and long-term potential for graft-derived benefits. The observed rejection response was characteristic of first-order allograft rejection and underscores a lack of immunological privilege in the microenvironment of the injured spinal cord.

Forelimb motor performance following dorsal column, dorsolateral funiculi, or ventrolateral funiculi lesions of the cervical spinal cord in the rat.

The neuroanatomical basis of forelimb motor control was examined following various surgical spinal cord lesions in the rat. Focal myelotomies were made at spinal level C4 to determine the effects that damage to long-tract pathways in the dorsal columns, dorsolateral funiculi, and ventrolateral funiculi have on a forelimb reaching and pellet retrieval task. Dorsal column lesions did not significantly reduce retrieval performance but did yield: (i) qualitative alterations in digit use during grasp execution, (ii) targeting errors during reaching attempts, and (iii) an apparent lack of ability to sense the presence of a pellet in the paw. Damage to the dorsolateral funiculi produced significantly diminished pellet retrieval performance at all postlesion intervals due to a prominent grasp deficit involving impaired digit flexion. Lesions of the ventrolateral funiculi did not produce a sustained, significant reduction in retrieval performance, although a qualitative deficit characterized by a mild forelimb reaching hypometria and premature grasp execution was exhibited. Based on comparisons with previous supraspinal and peripheral lesion studies in rats and supraspinal and spinal lesion studies in other mammalian species, the current results indicate that organization of descending and ascending spinal long-tract motor control of the forelimb in the rat is very similar to that described in other mammals, including primates. Additionally, these results demonstrate that the rat can serve as a biomedically relevant model of behavioral impairment and recovery following cervical spinal cord injury.

Forelimb motor performance following cervical spinal cord contusion injury in the rat.

The purpose of this study was to examine the degree, persistence, and nature of forelimb behavioral deficits following cervical spinal cord contusion injury in the rat. Forelimb reaching and pellet retrieval, forehead adhesive sticker removal, and vibrissae-induced forelimb placing were examined for 16 weeks following a weight-drop injury (10.0 g-2.5 cm) at the C4-C5 spinal level. Nine of 13 rats studied were unable to perform the pellet retrieval task due to pronounced forelimb extension hypometria. However, these animals did carry out the forehead sticker removal and vibrissae-induced placing tasks. Therefore, the loss of reaching ability related to pellet retrieval was not due to generalized paralysis. This interpretation was further supported by evaluation of the rostrocaudal extent of relative motoneuron loss from 1-mm divisions through the lesion zone. The extent of motoneuron pathology ranged from 2 to 6 mm but was largely confined to the C4-C5 spinal segments. Morphometric assessments of axonal sparing revealed that pellet retrieval performance during the last month of observation was significantly correlated with fiber sparing in the dorsal columns and ventral white matter, whereas no significant correlation could be demonstrated with regard to dorsolateral white matter. While there were no conspicuous differences in qualitative assessments of damage to interneuron pools (i.e., laminae V to VII) between the nonreaching and retrieval-recovered rats, the possibility of combined white and gray matter pathology contributing to this deficit still exists. These initial findings thus demonstrate that the weight-drop contusion injury model can be adopted to studies of cervical spinal cord trauma in the rat. Such lesions yield permanent deficits in forelimb function lending to future studies of possible therapeutic interventions. Furthermore, performance deficits observed at 1 week postinjury in the placing and forehead sticker removal tasks can be predictive of any potential for long-range spontaneous recovery in pellet retrieval ability.