Transplants of fibroblasts expressing BDNF and NT-3 promote recovery of bladder and hindlimb function following spinal contusion injury in rats

Abstract

We examined whether fibroblasts, genetically modified to express BDNF and NT-3 (Fb-BDNF/NT3) and transplanted into a thoracic spinal injury site, would enhance recovery of bladder function and whether this treatment would be associated with reorganization of lumbosacral spinal circuits implicated in bladder function. Rats received modified-moderate contusion injuries at T8/9, and 9 days later, Fb-BDNF/NT3 or unmodified fibroblasts (OP-controls) were delivered into the cord. Fb-BDNF/NT3 rats recovered from areflexic bladder earlier, showed decreased micturition pressure and fewer episodes of detrusor hyperreflexia, compared to OP-controls. There were also improvements in hindlimb function in the Fb-BDNF/NT3 group although locomotion on a more challenging substrate (grid) and tail withdrawal latency in response to a thermal stimulus showed persisting deficits, little recovery, and no differences between the groups. Immunocytochemistry at L6-S1 revealed changes in density of afferent and descending projections to L6-S1 cord. The density of small dorsal root axons increased in the superficial layers of the dorsal horn in OP-controls but not in Fb-BDNF/NT3, suggesting sprouting of primary afferents following injury that was inhibited by Fb-BDNF/NT-3. In contrast, the trophic factor secreting transplants stimulated sprouting and/or sparing of descending modulatory pathways projecting to the lumbosacral spinal cord. No differences in synaptophysin immunoreactivity were seen in the dorsal horn which suggested that synaptic density was similar but achieved by sprouting of different systems in the two operated groups. Fb-BDNF/NT3 transplanted into injured spinal cord thus improved both bladder and hindlimb function, and this was associated with reorganization of spinal circuitry.

Introduction

Autonomic dysfunction, which commonly accompanies severe spinal cord injury (SCI), is difficult to manage clinically and contributes to a poor quality of life (Hicken et al., 2001, Widerstrom-Noga et al., 2004). Following spinal injury, a dyssynergia between bladder and urethral sphincter (DSD) develops, leading to functional bladder outlet obstruction. This is identified by urinary retention and increased micturition pressure. Detrusor hyperreflexia (DHR) is seen as phasic bladder contractions during urine storage and results in urinary incontinence and high intravesical pressures, leading to bladder hypertrophy and deterioration of the upper urinary tract. Even modest increases in bladder pressure can elicit episodes of autonomic dysreflexia that can be life-threatening in SCI patients (Santajuliana et al., 1995). While there are some treatments for controlling DHR, for example, antimuscarinic agents (O'Leary et al., 2003, Pannek et al., 2000) and neurotoxins (capsaicin and resiniferatoxin) (Chancellor and de Groat, 1999, de Seze et al., 1999), DSD remains difficult to manage without catheterization or surgical interventions.

The functions of the lower urinary tract depend on neural circuits that are located in the lumbosacral cord. These include the bladder afferents projecting to the dorsal horn (DH), and the somatic dorsal lateral motor (DL) and spinal parasympathetic (SPN) nuclei that coordinate the activity of the urinary bladder, urethra, and urethral sphincter. These regions of lumbosacral cord are innervated by descending pathways from the brainstem, among which are axons containing corticotrophin releasing factor (CRF) and serotonergic and noradrenergic axons. Spinal injury above the lumbosacral level damages descending pathways and alters primary afferent pathways to the lumbosacral cord, thus impairing lower urinary tract function.

Reorganization of pathways caudal to a spinal lesion by sprouting, unmasking, or other compensatory mechanisms may contribute to the dysfunction that develops following severe injuries (de Groat et al., 1990, de Groat et al., 1998, Krenz et al., 1999), as well as to the partial recovery that occurs (de Groat et al., 1990, de Groat et al., 1998, Pikov and Wrathall, 2001). Transplant strategies can promote repair of SCI by selective modifications (regeneration and/or sprouting) of descending and primary afferent pathways and by their neuroprotective properties. Cellular transplants, if they survive and fill the cavity, have the potential to provide a substrate permissive for axonal growth while cells genetically modified to secrete neurotrophic factors may be effective in stimulating axonal growth and in providing neuroprotection, thus promoting recovery. The effects of these transplants may extend beyond the local circuitry since recovery of hindlimb function can be associated with cellular transplants at cervical (Kim et al., 2001, Shumsky et al., 2003) or thoracic (Coumans et al., 2001) sites of injury. The anatomical basis for repair and recovery is likely to include mechanisms of regeneration, sprouting, and neuroprotection that induce development of new relays or preserve damaged circuits and may therefore allow modifications in spinal interneuronal circuits (Bareyre et al., 2004, Hains et al., 2003, Vera and Nadelhaft, 2000).

Our aim was to determine whether transplants of fibroblasts genetically modified to produce BDNF and NT-3 (Fb-BDNF/NT3) would promote storage (reduce DHR) and permit efficient voiding at low pressures (amelioration of DSD) after spinal injury (Mitsui et al., 2003, Seki et al., 2002). We transplanted cells 9 days after the contusion injury as we have shown this to be associated with transplant mediated recovery in a different model (Mitsui et al., 2003) and because a delayed transplant paradigm may be more clinically relevant. Ideally, the treatments for bladder dysfunction would also improve motor function and diminish, or at least not exacerbate, neuropathic pain. We therefore tested motor (BBB, weight support during exploratory rearing) and sensorimotor (grid walking, withdrawal to noxious stimulus) functions. To evaluate changes in projections of pathways implicated in bladder control, we used immunocytochemistry to examine descending modulatory pathways and afferent pathways to the lumbosacral spinal cord. We found that Fb-BDNF/NT3 transplants promoted partial recovery of bladder and hindlimb motor function and that this improvement was associated with increased density of innervation by descending axons and diminished sprouting by primary afferent projections to the lumbosacral spinal cord.