RetroDISCO: Clearing technique to improve quantification of retrograde labeled motor neurons of intact mouse spinal cords.

BACKGROUND: Quantification of the number of axons reinnervating a target organ is often used to assess regeneration after peripheral nerve repair, but because of axonal branching, this method can overestimate the number of motor neurons regenerating across an injury. Current methods to count the number of regenerated motor neurons include retrograde labeling followed by cryosectioning and counting labeled motor neuron cell bodies, however, the process of sectioning introduces error from potential double counting of cells in adjacent sections. NEW METHOD: We describe a method, retroDISCO, that optically clears whole mouse spinal cord without loss of fluorescent signal to allow imaging of retrograde labeled motor neurons using confocal microscopy. RESULTS: Complete optical clearing of spinal cords takes four hours and confocal microscopy can obtain z-stacks of labeled motor neuron pools within 3-5min. The technique is able to detect anticipated differences in motor neuron number after cross-suture and conduit repair compared to intact mice and is highly repeatable. COMPARISON WITH EXISTING METHOD: RetroDISCO is inexpensive, simple, robust and uses commonly available microscopy techniques to determine the number of motor neurons extending axons across an injury site, avoiding the need for labor-intensive cryosectioning and potential double counting of motor neuron cell bodies in adjacent sections. CONCLUSIONS: RetroDISCO allows rapid quantification of the degree of reinnervation without the confounding produced by axonal sprouting.

Hyaluronan tetrasaccharide promotes regeneration of peripheral nerve: in vivo analysis by film model method.

Hyaluronan (HA) is known to inhibit neurons from regenerating in the central nervous system. However, hyaluronan tetrasaccharide (HA4) was found in in vitro experiments to promote outgrowth of neurons. To investigate the promotion by HA4 of nerve regeneration in vivo, we analyzed outgrowth of regenerating axons treated with HA4, using a film model method. After the common peroneal nerve in mice was transected, the proximal end of cut nerve was placed on a sheet of thin plastic film, immersed in several drops of HA4 solution, covered with another sheet of film, and then kept in vivo. Six hours after the procedure, terminal sprouts had grown out from ending bulbs formed at the cut end of parent nerve administered with HA4 solution 100 or 1000 µg/mL, while no sprouts were observed in groups treated with 10 µg/mL of HA4 or in controls. On the 2nd day after axotomy (day 2), many regenerating axons in the group treated with 100 µg/mL of HA4 extended onto the flat film for a longer distance than those treated with 1000 µg/mL of HA4 and controls. With the optimal dose of HA4 (100 µg/mL), axonal outgrowth was significantly (p<0.01) greater than that in controls at each time point. Schwann cells appeared migrating from parent nerve onto the film from day 3 as well as in controls. Thus, enhanced outgrowth of regenerating axons and normal behavior of migratory Schwann cells suggested that HA4 promoted regeneration of neurons without the mediation of Schwann cells.

Automatic image analysis of the postnatal growth of axons and myelin sheaths in the tibial and peroneal nerves of the rabbit.

The numerous morphometric studies on the myelinated fibers in the peripheral nerves have presented varying results. Only a few studies deal with the peripheral nerves from rabbits. In this work, a morphometric study was carried out on the tibial and peroneal nerves of new-born, 10-, 15-, 20-, 30-, 60-, 90- and 240-day-old rabbits. The bilateral proximal segments of both nerves were investigated. Negatives of semi-thin cross sections were used for myelinated fiber morphometric analysis, carried out by an OLYMPUS Video image analyser. Two morphometric parameters, the average axon diameter (AD) (the average length of Feret's diameters) and the specific width of the myelin sheaths (SWMS) (specifying the total width of the myelin sheath), were evaluated for every age group. In the tibial and peroneal nerves a bimodal distribution of the average AD appeared on the 20th day, and of the SWMS on the 10th day postnatum. A tight correlation was obtained when comparing mean AD (mAD) and mean SWMS (mSWMS) in new-born and 240-day-old rabbits. From birth to adulthood the mAD increased in both nerves by about 270% and the mSWMS by about 280%. The mAD/mSWMS ratio in both new-born and 8-month-old rabbits was found to be 4 in the tibial and 5 in the peroneal nerves. According to the available data, an approach to the measurement of AD as an average length of Feret's diameters and the measurement of the myelin thickness as a SWMS has not yet been employed. An extension of this methodological approach could help to understand the growth and myelinization of peripheral nerve fibers.

Permanent alterations of spinal cord reflexes following nerve lesion in newborn rats.

Sciatic nerve lesion in newborn rats is known to cause degeneration of a large number of axotomized motoneurones and spinal ganglion cells. Some of the surviving motoneurones exhibit abnormal firing properties and the projection pattern of central terminals of sensory neurones is altered. We report here on long-term changes in spinal cord reflexes in adult rats following neonatal nerve crush. In acutely spinalized and anaesthetized adult rats 4-6 months old in which the sciatic nerve had been crushed on one side at birth, the tibial nerve, common peroneal nerve or sural nerve were stimulated on the reinnervated and control side and reflex responses were recorded from the L5 ventral spinal roots. Ventral root responses (VRRs) to tibial and peroneal nerve stimulation on the side of the nerve lesion were significantly smaller in amplitude representing only about 15% of the mean amplitude of VRRs on the control side. The calculated central delay of the first, presumably monosynaptic component of the VRR potential was 1.6 ms on the control side while the earliest VRR wave on the side of the nerve lesion appeared after a mean central latency of 4.0 ms that seems too long to be of monosynaptic origin. These results suggest that neonatal sciatic nerve injury markedly alters the physiological properties and synaptic connectivity in spinal cord neurones and causes a marked depression of spinal cord responses to peripheral nerve stimulation.

Persistence of somatic and dendritic growth associated processes and induction of dendritic sprouting in motoneurones after neonatal axotomy in the rat.

The effect of neonatal axotomy on the maturation of motoneurone somadendritic morphology was studied in identified motoneurones innervating the ankle dorsiflexor muscles tibialis anterior (TA) and extensor digitorum longus (EDL) of the rat by intracellular injection of Lucifer Yellow and confocal microscopy. At birth, the entire somatodendritic surface is covered with fine filopodial growth-associated processes. These are eliminated from the soma and proximal dendrites during the first postnatal week as part of a somatofugal process of dendritic maturation. Following neonatal axotomy, the postnatal elimination of growth associated processes was halted and new, axonal-like processes were seen to sprout from the soma and proximal dendrites in some of the axotomized motoneurones. These results indicate that synaptic interaction with the target muscle during the early postnatal period is essential for the maturation of the somatodendritic receptive surface of the motoneurone.

Effects of predegeneration on nerve regeneration through silicone Y-chambers.

The effects of nerve predegeneration on the preferential growth of regenerating axons were studied using a silicone Y-chamber model. This system provided a choice for axons to grow towards two distal nerve options, either a 7-day predegenerated nerve segment (PNS) or a fresh nerve segment (FNS). The rat peroneal or tibial nerve was inserted into the proximal intlet and the PNS and FNS of the corresponding nerve were inserted into the distal outlets. At 28 days postoperative, the size of the distal regenerate was significantly greater (26%) towards the PNS for the tibial nerve group. The density and number of regenerated myelinated axons in the distal nerve segment was greater on the PNS for both the tibial (97 and 88%, respectively) and peroneal (221 and 221%, respectively) nerve groups. In contrast, the elevated density and number of nonvascular nuclei was relatively constant for both PNS and FNS. Immunocytochemical and ultrastructural evidence support the hypothesis that the early activation of Schwann cells is primarily responsible for the enhanced regeneration and maturation observed in PNS. It is suggested that PNS might improve the outcome after clinical repair of injured peripheral nerves.

A sensitive period in gestation for nicotine acceleration of neuromuscular maturation.

Nicotine, administered to pregnant Sprague-Dawley rat dams during gestation, altered the maturation of the developing extensor digitorum longus (EDL) muscle-peroneal nerve complex of the 2-week-old offspring. Initial isometric twitch time to peak and rate of rise of tension and tetanus time to peak tension of the group treated with nicotine during gestational (G) days G3-G8 were faster than controls indicating accelerated maturation of EDL muscle. Contractile parameters of the group treated with nicotine during G9-G13, a period immediately prior to muscle innervation, differed little from control. Nicotine administered during G14-G21 significantly increased twitch and tetanus tension and twitch rate of rise. There was little to no effect on motor unit size or number of motor units with nicotine during any of the three prenatal periods. Thus, while late prenatal nicotine administration primarily altered the strength of muscle, early gestational administration of nicotine increased the rate of muscle development suggesting a possible sensitive period for the accelerative action of nicotine on muscle maturation at this time.

Postnatal development of peroneal motoneurons in the kitten.

In 1- to 72-day-old kittens, motoneurons of the 3 peroneal muscle nuclei were labeled by retrograde axonal transport of horseradish peroxidase from individual muscles. At birth, the locations of peroneal nuclei were similar to those of the adult cat. Counts of motoneurons at different ages indicated that postnatal cell death does not occur in peroneal motor nuclei. Primary dendrites were as numerous in motoneurons of newborn kittens as in adult motoneurons but they were thinner, shorter and poorly ramified. The number of recurrent axon collaterals was higher in the first postnatal week than at later stages. The growth of motoneurons followed similar rates in the 3 peroneal nuclei. Distributions of cell body diameters and volumes were unimodal at birth and became bimodal between 15 and 20 days postnatal. The separation of peroneal motoneurons in two size subgroups, presumably corresponding to alpha and gamma populations, was followed by an increase in growth rate which became faster for alpha than for gamma motoneurons.

Mammalian neuromuscular development accelerated with early but slowed with late gestational administration of ACTH peptide.

The neuropeptide ACTH 4-10, a nonsteroidogenic fragment of adrenocorticotropic hormone, has two distinct and opposite effects on developing nerve and muscle. Muscle is positively influenced by ACTH during the first part of gestation (G days 3-12) before innervation occurs. Subsequent effects on innervation are largely depressive and exerted only during G13-21. Treatment during G3-12 increases twitch amplitude, rise time and speed of contraction of directly and indirectly stimulated extensor digitorum longus (EDL) muscle of two wk old rats. Treatment during G13-21 slows contractions of indirectly stimulated EDL, whereas treatment throughout gestation (G3-G21) shows little effect. Thus, ACTH first accelerates muscle development then modulates this development through neuronal depression.