Esbjörn Bergman

Factors contributing to neuromuscular impairment and sarcopenia during aging.

Motor disturbances and wasting of skeletal muscles (sarcopenia) causes significant impairment of daily life activities and is a major underlying cause for hospitalization in senescence. Herein we review data and present new findings on aging-specific changes in motoneurons, skeletal muscle and the interplay between motoneurons and target muscle fibers. Although many of the changes occurring during aging may be specific to motoneurons and myofibers, respectively, evidence indicates that myofiber regeneration in sarcopenic muscle is halted at the point where reinnervation is critical for the final differentiation into mature myofibers. Combined, evidence suggests that sarcopenia to a significant extent depend on a decreased capacity among motoneurons to innervate regenerating fibers. There are also conspicuous changes in the expression of several cytokines known to play important roles in establishing and maintaining neuromuscular connectivity during development and adulthood. We also present data showing the usefulness of rodent models in studies of successful and unsuccessful patterns of aging. Finally, we show that not only dietary restriction (DR) but also activity and social environment may modulate the pattern of aging.

Loss of primary sensory neurons in the very old rat: neuron number estimates using the disector method and confocal optical sectioning.

Loss of neurons has been considered to be a prime cause of nervous disturbances that occur with advancing age. However, the notion of a constitutive aging‐related loss of neurons has been challenged recently in several studies that used up‐to‐date methods for counting neurons. In this study, we have applied stereological techniques with the objective of obtaining quantitative data on total neuron numbers and the distribution of neuron cross‐sectional areas in the fifth cervical (C5) and fourth lumbar (L4) dorsal root ganglion (DRG) of 3‐ and 30‐month‐old Sprague‐Dawley rats. Tissue data were recorded on a confocal laser‐scanning microscope with the use of the optical‐disector technique and random, systematic sampling.

Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging.

We have here sought to cross-correlate the expression of immunoreactivities for several neuropeptides, nitric oxide synthase (NOS) and the growth associated protein GAP-43 in subpopulations of dorsal root ganglion (DRG) neurons tagged by the selective markers isolectin B4 and the neurofilament antibody RT97, selective for, respectively, subpopulations of small and large DRG neurons. By use of double- and triple-labeling immunohistochemistry, non-manipulated and sciatic nerve transected young adult rats as well as aged (30-months-old) rats were examined using a confocal microscope equipped with enhanced spectral separation. In young adult rats, the DRG neuron profiles could be divided into three subpopulations (B4 binding (B4+) approximately 50%; RT97-immunoreactive (RT97+) approximately 35%; B4-/RT97- approximately 15%). Calcitonin gene-related peptide (CGRP) is expressed in all three subpopulations. Galanin message-associated peptide (GMAP) colocalize with CGRP (100%) but is not expressed in RT97+ profiles. NOS is present in the RT97- subpopulations and frequently colocalize with CGRP (92%). GAP-43 is expressed in all three DRG subpopulations and colocalize with CGRP (88%), GMAP (38%) and/or NOS (22%). Only very small differences were seen among the young adult rats, implicating that the size of respective subpopulation as well as the expression pattern for neuropeptides, NOS and GAP-43 are fairly stable. Sciatic nerve transection reduced B4-binding but not RT97-like immunoreactivity. Distinct changes in the expression of neuropeptides, NOS and GAP-43 were evident in the DRG subpopulations and, furthermore, the regulatory changes were very similar among the lesioned animals. The relative size of the DRG subpopulations was unaffected by aging, while the expression of neuropeptides was altered showing similarities with the changes induced by axotomy in young adult rats.

Neuropeptides and neurotrophin receptor mRNAs primary sensory neurons of aged rats

Neuropeptides and neurotrophin receptors are regulated in primary sensory neurons in response to axonal injury, and axonal lesions are characteristic stigmata of aging primary sensory neurons. We have therefore examined the expression of neuropeptides and neurotrophin receptor mRNAs in 30‐month‐old (median survival age) Sprague‐Dawley rats to see if similar adaptive mechanisms operate in senescence. The content of neuropeptides was examined with immunohistochemistry (IHC) and in situ hybridization (ISH), and the cellular mRNA expression of neurotrophin receptors was studied with ISH. All of the aged rats had symptoms of hind limb incapacity (posterior paralysis), but fore limbs did not seem affected. The size‐distribution of neuronal profiles in cervical and lumbar dorsal root ganglia (DRGs) was similar in aged and young adult (2–3 months old) rats. In aged rats, the DRG neurons showed an increase in both immunolabelling and mRNA content of neuropeptide tyrosine (NPY), as well as an increased cellular expression of galanin mRNA. In the same animals, there were decreased cellular levels of calcitonin gene‐related peptide (CGRP; IHC and ISH) and substance P (SP; IHC and ISH), while the difference in neuronal somatostatin (IHC and ISH) was small. The distribution of neuropeptide immunoreactivities in the dorsal horn of the corresponding spinal cord segments revealed a decreased labelling for CGRP‐, SP‐, and somatostatin‐like immunoreactivities (LI) in the aged rats at both cervical and lumbar levels. NPY‐ and galanin‐LI had a similar distribution in aged and young adult rats. NPY‐immunoreactive fibers were also encountered in the dorsal column of aged but not young adult rats. ISH revealed that most of the primary sensory neurons express mRNA for the p75 low‐affinity neurotrophin receptor (p75‐LANR) and that there was no discernible difference between young adult and aged rats. The labelling intensity for mRNA encoding high‐affinity tyrosine kinase receptors (TrkA, TrkB, and TrkC) was decreased in aged rat DRG neurons, while the percentage of neuronal profiles expressing mRNA for TrkA/B/C was similar in young adult and aged rats. The changed pattern of neuropeptide expression in primary sensory neurons of aged rats resembled that seen in young adult rats subjected to axonal injury of peripheral sensory nerves and may, thus, indicate aging‐related lesions of sensory fibers. Since NPY is primarily present in large and galanin in small DRG neurons, the stronger effect on NPY as compared to galanin expression may indicate that aging preferentially affects neurons associated with mechanoreception (Aα and Aβ fibers) as compared to nociceptive units (A and C fibers). Furthermore, the observed changes in neuropeptide expression were most pronounced in lumbar DRGs, that harbors the sensory neurons supplying the affected hindlimbs of the rats.

EFFECTS OF AGING AND AXOTOMY ON THE EXPRESSION OF NEUROTROPHIN RECEPTORS IN PRIMARY SENSORY NEURONS

Aging is accompanied by declined sensory perception, paralleled by widespread dystrophic and degenerative changes in both central and peripheral sensory pathways. Several lines of evidence indicate that neurotrophic interactions are of importance for a maintained plasticity in the adult and aging nervous system, and that changes in the expression of neuro‐trophins and/or their receptors may underpin senile neurodegeneration. We have here examined the expression of neurotrophin receptor (p75NTR, trkA, trkB, and trkC) mRNA and protein in intact and axotomized primary sensory neurons of young adult (3 months) and aged (30 months) rats. To examine possible differences among primary sensory neuron populations, we have studied trigeminal ganglia (TG) as well as cervical and lumbar dorsal root ganglia (DRG). In intact aged rats, a decrease in trk (A/B/C) mRNA labeling densities and protein‐like immuno‐reactivities was observed. The decrease was most pronounced in lumbar DRG. In contrast, a small, not statistically significant, increase of p75NTR expression was observed in aged DRG neuron profiles. After axotomy, a down‐regulation of mRNA and protein levels was observed for all neurotrophin receptors (p75NTR, trkA, trkB and trkC) in both young adult and aged rats. Consistent with the higher expression levels of neurotrophin receptors in unlesioned young adult primary sensory neurons, the relative effect of axotomy was more pronounced in the young adult than aged rats. Although a decrease in mean cell profile cross‐sectional areas was found during aging and after axotomy, the characteristic distribution of neurotrophin receptor expression in different populations of NRG neurons was conserved. The present findings suggest an attenuation of neurotrophic signaling in primary sensory neurons with advancing age and that the expression of p75NTR and trks is regulated differently during aging. A similar dissociation of p75NTR and trk regulation has previously been reported in other neuronal systems during aging, suggesting that there may be a common underlying mechanism. Decreased access to ligands, disturbed axon function and systemic changes in androgen/estrogen levels are discussed as inducing and/or contributing factors. J. Comp. Neurol. 410:368–386, 1999.

Reciprocal changes in the expression of neurotrophin mRNAs in target tissues and peripheral nerves of aged rats.

trk receptors are downregulated in both dorsal root ganglion (DRG) and spinal motoneurons of aged rats with behavioral sensorimotor deficits. Here we provide evidence, using reverse transcription-polymerase chain reaction (RT-PCR), of decreased levels of neurotrophin (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3; and neurotrophin-4, NT-4) mRNAs in target muscles. Moreover, the degree of neurotrophin mRNA decrease in target muscles seems to co-vary with the extent of sensorimotor disturbances. In contrast, the peripheral nerve of aged rats showed a reciprocal regulation of neurotrophins, with increased levels of NGF, BDNF, and NT-4 mRNAs. Taken together, evidence suggest an aging-related attenuation of neurotrophin signaling between target tissues, on one hand, and DRG and motoneurons, on the other, and, furthermore, that target-derived neurotrophins regulate the expression levels of trk mRNAs in both DRG and motoneurons.

Iron load and redox stress in skeletal muscle of aged rats.

Loss of skeletal muscle mass (sarcopenia) is a major contributor to disability in old age. We used two‐dimensional gel electrophoresis and mass spectrometry to screen for changes in proteins, and cDNA profiling to assess transcriptional regulations in the gastrocnemius muscle of adult (4 months) and aged (30 months) male Sprague‐Dawley rats. Thirty‐five proteins were differentially expressed in aged muscle. Proteins and mRNA transcripts involved in redox homeostasis and iron load were increased, representing novel components that were previously not associated with sarcopenia. Tissue iron levels were elevated in senescence, paralleling an increase in transferrin. Proteins involved in redox homeostasis showed a complex pattern of changes with increased SOD1 and decreased SOD2. These results suggest that an elevated iron load is a significant component of sarcopenia with the potential to be exploited clinically, and that mitochondria of aged striated muscle may be more vulnerable to radicals produced in cell respiration. Muscle Nerve, 2007

Effect of a graded single constriction of the rat sciatic nerve on pain behavior and expression of immunoreactive NPY and NPY Y1 receptor in DRG neurons and spinal cord

In the present study, the rat sciatic nerve was constricted to varying degrees using only one ligature with a very thin polyethylene sheath placed between nerve and ligature thread. Complete nerve transection was studied for comparison. With a 40-80% constriction of the nerve we observed allodynia to a similar extent as in the so-called Bennett model based on four loose ligatures. We also monitored changes in the expression of neuropeptide Y (NPY) and the NPY Y1 receptor (Y1R) in the lumbar 4-5 dorsal root ganglia (DRG) and dorsal horn and found upregulation of NPY and downregulation of the Y1R in DRG neurons after injury. These results indicate that similar peptide and receptor changes occur in this model as after axotomy and in other nerve injury models, although the immunohistochemical and behavioral changes seem to be dependent on the degree of constriction of the nerve. Thus, it seems relevant to monitor the degree of constriction when evaluating pain and other post-injury events. The possibility that some of the changes in NPY-ergic neurotransmission are related to the generation of allodynia is discussed; as well as the possibility to use this mononeuropathic model based on a single ligature nerve constriction (SLNC) as a complementary approach to other widely used pain models.

Regulation of NGF-family ligands and receptors in adulthood and senescence: correlation to degenerative and regenerative changes in cutaneous innervation

During development, a highly differential neurotrophin dependency is reported for various types of nerve endings in the whisker follicle. To what extent these dependencies extend and play a role in adulthood is largely unresolved. We show here, using in situ hybridization and immunohistochemistry that the expression of neurotrophins and trk/p75 receptors persists in adulthood. As suggested by their expression profiles, many classes of cutaneous nerve endings disclose similar ligand–receptor dependencies in adult animals as during development, while other populations appear to switch their dependency. Furthermore, our data suggest that sensory endings that have a high turnover due to mechanical wear and tear, e.g. Merkel cell–neurite complexes at the level of ring sinus show a more complex ligand–receptor expression phenotype than do endings with a less vulnerable location, e.g. the Merkel cell–neurite complexes at the rete ridge collar. Thus, neurotrophin‐3 (NT3)/trkA signalling is suggested to be important for a continuous terminal plasticity of Merkel cell–neurite complexes at the level of ring sinus in adulthood. Evidence supporting a role for neurotrophin signalling in maintaining the adult cutaneous innervation also comes from the close correlation between altered ligand–receptor expression(s) and axonal/terminal aberrations in senescence. Thus, an ageing‐related decrease in target neurotrophin expression, notably NT3 and NT4, results in a site‐specific loss of sensory terminals concomitant with an aberrant growth of regenerating/sprouting axons into new target fields. Ageing of the cutaneous innervation, manifested in degenerative and regenerative events, seems strongly associated with changes in neurotrophic interactions between sensory neurons and target tissues.

Regulation of neurotrophin signaling in aging sensory and motoneurons

A hallmark of senescence is sensorimotor impairment, involving locomotion and postural control as well as fine-tuned movements. Sensory and motoneurons are not lost to any significant degree with advancing age, but do show characteristic changes in gene-expression pattern, morphology, and connectivity. This review covers recent experimental findings corroborating that alterations in trophic signaling may induce several of the phenotypic changes seen in primary sensory and motoneurons during aging. Furthermore, the data suggests that target failure, and/or breakdown of neuron-target interaction, is a critical event in the aging process of sensory and motoneurons.