Weight-bearing locomotion in the developing opossum, Monodelphis domestica following spinal transection: remodeling of neuronal circuits caudal to lesion

Abstract

Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum Monodelphis domestica whose young are born very immature, allowing access to developmental stages only accessible in utero in eutherian mammals. Spinal cords of Monodelphis pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors' gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.

Figure 1. Behavioural assessments.

Control (n = 11, white bars), P7-injured (n = 7, black bars) and P28-injured (n = 9, grey bars) opossums. A: BBB Locomotor rating scores. B: Representative gait traces from treadmill locomotion. C: Regularity index. D: Step duration. E: Stance duration. F: Swing duration. Mean ± sem; *P≤0.05 vs control; ^# P≤0.05 vs P7-inj, by One-way ANOVA.

Figure 2. Limb placement phase lags.

Control (n = 11, white bars), P7-injured (n = 7, black bars) and P28-injured (n = 9,grey bars) opossums. A: Schematic examples showing calculation method for girdle, diagonal and ipsilateral phase lags. These examples are taken from the control animal in Fig. 1B. B: Forelimb and hindlimb girdle phase lags C: Diagonal phase lags. D: Ipsilateral phase lags. Mean ± sem; *P≤0.05 vs control; ^# P≤0.05 vs P7-inj, by One-way ANOVA.

Figure 3. Morphometric measurements of Monodelphis spinal cords.

Control (n = 5, white circles), P7-injured (n = 3, black circles) and P28-injured (n = 4, grey circles) opossum spinal cords. A: Representative whole mounts of lower cervical and thoracic spinal cords from control (top), P7-injured (middle) and P28-injured (bottom) Monodelphis. B: Whole spinal cord cross-sectional area along the length of the spinal cord. C: Grey matter area. D: White matter area. Mean ± sem. *P≤0.05 vs control. Stars denoting significance appear immediately above data for P7-injured animals or immediately below data for P28-injured animals.

Figure 4. Propriospinal labelling in the spinal cord.

A : Schematic of labelling protocol and areas of interest. B: Labelled neurons in spinal cord of control (white bars), P7-injured (black bars) and P28-injured (grey bars). C : Representative images of labelled neurons in the lower spinal cord. Note the lack of labelling rostral to the lesion in the P28-injured cord, indicating that no axons from propriospinal neurons crossed the lesion site. D : 2D reconstruction of labelling in the L1–2 spinal segments. This was the level at which marker was injected. The extent of they dye in the ispilateral cord is indicated; no neuron counts could be made in this area because of the presence of injected fluorescent marker. E : 2D reconstruction of labelling in the L3–5 spinal segments. Mean ± sem; *P≤0.05 vs Control, by One-way ANOVA.

Figure 5. Gene expression quantitation by qRT-PCR for L1–2 and L3–5 spinal segments.

Gene expression changes in the spinal cords of P7- (solid bars) and P28-injured (hatched bars) opossums are shown relative to control expression. A: Gene expression ratios for the L1–2 spinal segments. B: Gene expression ratios for the L3–5 spinal segments. All data are mean ± sem. *P≤0.05; P values ≤0.1 are indicated in parentheses.

Figure 6. Re-transection of P28-injured opossum spinal cord.

In adulthood the spinal cord of one P28-injured Monodelphis was re-transected through the centre of the injury site and allowed to recover for 3 weeks before behavioural and labelling studies were performed. A : Representative gait trace from P28-injured opossum in adulthood before (left panel) and after (right panel) re-transection. B : Image of section through the medulla of re-transected P28-injured animal following retrograde tracing. C : Images of sections through the spinal cord above (left panel) and below (right panel) the level of the re-transection. Re-transection was performed at the same level as the initial transection during the neonatal period and did not result in a second distinct transection site (data not shown).