Transplants of fibroblasts expressing BDNF and NT-3 promote recovery of bladder and hindlimb function following spinal contusion injury in rats
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.
Section snippets
Animal groups
Twenty-nine female Sprague–Dawley rats (225–250 g; Taconic, Germantown, USA) were studied. Twenty-three rats received a modified-moderate contusion. Nine days later, unmodified fibroblasts (OP-controls, n = 11) or a mix of fibroblasts genetically modified to produce BDNF and NT-3 (Fb-BDNF/NT3, n = 12) were injected into the injured spinal cord. All rats were tested for open field locomotion (BBB) and cystometry. Rats were randomly divided into two subgroups for additional behavioral testing.
Micturition behavior assessed in metabolic cages
Both operated groups showed an initial period of bladder areflexia when manual expression was required to induce voiding. This deficit was followed by a partial recovery of micturition reflexes. ANOVA revealed a significant interaction between groups over time [F(7,63) = 2.1, P < 0.05]. Voided volume per micturition increased in the Fb-BDNF/NT3 group by 2 weeks following transplantation while OP-controls reached similar levels only by week 5 (Fig. 1). The increase in voiding volume/micturition
Recovery of lower urinary tract function
Micturition is mediated by spinal reflexes that are normally under descending control. Bladder dysfunction occurs after SCI because of the damage to descending pathways and alterations in primary afferent pathways that normally coordinate somatic motor and parasympathetic control of the lower urinary tract and reflex activity (Beattie et al., 1993). SCI induces a period of bladder areflexia that is followed by the slow development of involuntary reflex micturition and detrusor overactivity,
Acknowledgments
This study is supported by NS 24707, Spinal Cord Research Foundation (No. 2312-01) of the Paralyzed Veterans of America, United Spinal Association and Uehara Memorial Foundation. We gratefully acknowledge the input that our laboratories provided discussions and constructive criticism. We wish to thank William D. Long, Harra Sandrow, Nicole M. Amato, Carl Coleman, Andrew M Diamond, Katina C. Hanford, Maria A. Obrocka, and Theresa Connors for their excellent technical assistance. We thank Dr.
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2014, Tzu Chi Medical JournalCitation Excerpt :Repair of spinal cord trauma by nerve grafts and stabilization of the grafted area with fibrin glue containing acid fibroblast growth factor have been successful in treating chronic paraplegia [184]. Transplants of fibroblasts expressing brain-derived nerve growth factor and neurotrophin-3 have been applied in an animal SCI model and found to improve both bladder and hindlimb function, which was associated with reorganization of spinal circuitry [185]. Transplantation of immortalized neural stem cells into the injured spinal cord could promote recovery of voiding function in rats [186].