Effects of Age on Myenteric (and sensory and other) Neurons

1. Effects of Age on Myenteric (and sensory and other) Neurons Karen E. Hall, M.D., Ph.D. Ann Arbor VA Healthcare System University of Michigan Healthcare System

3. Animal models of aging vs human studies: heterogeneity in physiology Age range of “old” test subjects is important • Appears to be physiologic decline in “oldest-old” compared to “middle-old” or “young-old” • Rats: >28 months • Humans: > 85 years

4. Hall and Wiley, Hazzard, Principles of Geriatric Medicine, 1999

5. Overview • Aging vs disease: gut regions at risk • Physiology of neuronal aging: • Enteric nerves • Dorsal root ganglia • Other neurons • Vulnerability to injury with aging • Neurotrophin support

6. Clinical effects of Aging on GI Function Normal aging associated with: Slow transit of food bolus, UES dys-coordination Decreased LES pressure, acid clearance Decreased acid production, impaired mucosal blood flow Increased colonic pressure, slow colonic transit Upper and distal GI tract most vulnerable Dysphagia, GERD, impaired acid secretion, PUD Constipation, diarrhea, diverticuli

7. Small bowel is “protected” from aging • Minimal effect of aging on: • Small intestinal secretion, absorption, motility • Biliary secretion • Pancreatic exocrine function

8. Sensory changes with Aging Dorsal root ganglion neurons • Decreased sensation (somatic and visceral) • Loss of vibration, position sense in feet • Falls • Diminished peritoneal sensation with inflammation • Misdiagnosis of peritonitis • Impaired vascular response to capsaicin • Increased risk of mucosal ischemia/injury

9. Scott and Mertz Clin Perspectives Gastro 2001

10. Enteric Microcircuits 4. Excitatory motor neuron (ACh/Sub P) 3. Ascending neuron 2. ENS stimulated 1. Distention or mucosal stimulation releases 5-HT 5. Descending neuron 6. Inhibitory motor neuron (NO, VIP, ATP) Scott and Mertz Clin Perspect Gastro 2001

11. Effect of Aging on the ENS 37% decrease in myenteric and submucous plexus neurons in rats and humans(CGRP, ACh, VIP) Increased non-neuronal tissue • Santer and Baker J Auton Nerv 1988; Belai et al. Cell Tissue Res 1995; Gomes et al Gerontol 1997; Wade, AGA 2002 Gradient of neuronal loss in SB and colon: distal > proximal • Phillips and Powley J Comp Neurol 2001

12. Loss of myenteric CGRP+ neurons may impair contractility • CGRP: anterograde trophic agent • Co-localizes with ACh, promotes ACh receptor biosynthesis, increases contraction Loss of CGRP+ neurons also documented in sciatic nerves of Fisher 344 rats aged 24 mo Sciatic CGRP production 50% in aged rats Fernandez and Hodges-Savola Neurochem Res 1994

13. NOS expression/release increased in aged ENS Increased percentage of NOS-containing myenteric neurons in proximal colon in humans and rats Human tissue: pre-term fetuses (14-17 wks) Adult (50 y), aged (80 y), Crohn’s (30 y) NADPH-diaphorase Aged > fetal > Crohn’s > adult • Belai and Burnstock Dig Dis Sci 1999 Associated with decreased motility and slow transit in colon: effect may be additive to impaired Ach and Ca signaling

15. Sensation and Pain 1.Vagal and Sacral Afferent Neurons (Sub P, CGRP) Sensation, Pain, Vascular responses 2. Spinal Afferents (Dorsal Root Ganglia) (Sub P, CGRP) Scott and Mertz Clin Perspect Gastro 2001

16. Loss of DRG neurons in aging: controversial Older literature: decrease in large fibers (position and vibration) and corresponding soma in lumbosacral DRGs Recent reports: little or no change in number by serial reconstruction in rats Mohamned and Sauter Neurosci Lett 2001 Nuclear:cytoplasmic ratio, number of terminals Ledda et al. Neurosci Lett 2000

17. Extrinsic Secretory-Motor • Vagal Parasympathetic Efferent Neurons • Excitatory Sphincters: inhibitory 5. Sympathetic Efferent Neurons Inhibitory Sphincters: excitatory Scott and Mertz Clin Perspect Gastro 2001

18. Aging: Vagal and Sympathetic Innervation • UES relaxation delayed • Increased simultaneous esophageal contractions • Abnormal inhibitory ENS neurons (NO, VIP) implicated, vagal input less affected Smith and Mertz Clin Perspect Gastroenterol 2001

19. Aging: Vagal and Sympathetic Innervation • 22-62% loss of ENS neurons in upper esophagus • Meciano et al. Gerontol 1995 • Receptive relaxation of stomach (vago-vagal) not impaired

20. Decreased Vascular responses in Aging • Aged (12 mo) rats: mesenteric blood flow in SMA induced by capsaicin and 0.1 N HCl decreased by 50% vs. 2 mo rats • Seno et al. Dig Dis Sci 1996 **

21. Decreased GI Vascular responses in Aging CGRP implicated: release by electrical stimulation significantly decreased Decreased vasodilatory response to isoproteranol No effect of aging on vascular response to nitroglycerine, histamine, ACh • Li and Duckles Eur J Pharmacol 1993

22. Aging and neuronal calcium signaling • Decreased influx of calcium in ENS, DRGs: • Action potential (Na+) decreased • Decreased neurotransmitter release • Slow conduction • Decreased sensation, motility Sheng et al J Neurophysiol 2001, Roberts et al. J Pharm Exp Ther 1994

23. Aging and neuronal calcium signaling Cytosolic calcium [Ca2+]i release by K+, capsaicin decreased in ENS and sensory neurons: smaller peak [Ca2+]i response Impaired calcium re-uptake in storage pools (SER and mitochondrial) Buchholtz and Duckles Neurobiol Aging 1998

24. Ca2+-binding proteins increased in aging • Increased calretinin- and calbindin-immunoreactive neurons in proximal colon in aged humans and rats • Calbindin: Aged > fetal > Crohn’s > adult • Belai and Burnstock Dig Dis Sci 1999

25. Calcium hypothesis of neuronal injury Choi Nature Supp 2000

26. Aging and neuronal calcium signaling Is increased calbindin protective? Is decreased calcium influx also protective or is it a primary impairment of aging? Elevation of Ca influx by ionophores or by trophic factors (NGF) restores transmitter release to youthful levels

27. Effect of Aging on Axoplasmic Transport Decreased retrograde transport of neurotrophins to soma in vagal and DRG neurons • diminished production of NGF by target tissue • Slow transport: binding proteins, microtubules Decreased CGRP transport (both retrograde and antegrade)

28. Neurotrophin expression in the ENS Fetal • NT-3 and trk C involved in early development of ENS • NT-3 knockout mice: reduced CGRP+ interneurons (Chalazonitis 2002) Adult • BDNF, trk B in myenteric, submucous plexus, blood vessels • Trk C and NT-3 expression in submucous >> myenteric • Most trk C+ neurons contain VIP, not Sub P • Trk A low, but increased in IBD (DiMola et al Gut 2000)

29. Neurotrophins: role in gut motility Maintain circuitry • GDNF required for normal neural crest cell migration and ENS development • Decreased trk C and NT-3 in Hirschsprung’s disease modulate neurotransmission and peptide expression • BDNF and NT-3 increase small bowel and colonic transit: ameliorate constipation • Trk A knockouts and anti-trk A associated with loss of CGRP expression in DRGs and ENS • (Snider and Silas-Santigo Proc R Soc Lond 1996)

30. Vulnerability to Neuronal Injury in Aging Differences in response to axotomy in aged may be related to neurotrophin dependence: • Fetal neurons require neurotrophins for normal development (both survival and sculpturing) • Adult neurons: diminished requirement for neurotrophin support • Aged: Expression of neurotrophins and trk receptors decreased, survival impaired in-vitro

31. Trk A p75NTR Western blot of homogenized DRG ganglia from Young (6 mo) and Aged (28 mo) Fisher 344 x Brown Norway rats +NGF: NGF 100 ng/ml x 24 hours Sheng et al. J Neurophys 2001

32. NGF Treatment in-vitro rescues Aged DRG neurons Increased expression of trk A: correlated with significant increase in survival (40 to 75%) of cultured DRG neurons from aged animals x 96 hours Sheng et al. J Neurophys 2001

33. Neurotrophin signaling decreased in aging • Decreased trk A, B, C: mRNA and protein in lower thoracic and lumbar DRGs from aged (28-30 mo) rats • p75NTR unchanged • Bergman et al. J. Comp Neurol 1999 • Sheng et al. J Neurophys 2001

34. Response to Neuronal Injury in Aging • Axotomy: faster and more extensive axonal degeneration in young animals • Sciatic axotomy in aged rats (>24 mo): • Soma less likely to be lost following axotomy • Regeneration of axon is slower, all trk receptors decreased expression • No change in number of terminal processes, but decreased re-occupation of motor nerve terminals • Kerezoudi and Thomas Gerontol 1999

35. “Oxidative Stress” Peroxidation + decreased free radical scavengers (SOD, glutathione) • Mitochondrial function implicated: • Decreased ATP generation (depolarized mitochondrial membrane potential y) • Scavengers not reduced • Intracellular oxidation – impaired function

36. Neuronal Mitochondrial Impairment • Mitochondrial DNA deletions (mtDNA4834) 300 x increase in old (24 mo) vs young (6 mo) in myenteric neurons • Correlated with decreased vascular response to capsaicin • Nagley et al. Neurobiol Aging 2001

37. General Observations of Neuronal Aging In the GI Tract • ?Decreased neuronal number • Decreased axoplasmic transport of trophic factors and neurotransmitters • Slowing of nerve conduction • Increased susceptibility to damage by noxious insults but soma less likely to degenerate following axotomy • Improved survival and/or function with trophic support

38. Treatment: Can we teach an old dog new tricks? • Increase efficacy of existing pathways • Target metabolic changes (Ca, neurotransmitters) • Provide exogenous neurotrophins or equivalents • Prevent neuronal death • Replace old cells with new (?stem cells)

39. Acknowledgements: John Wiley Yu Genggeng Huaibao Sheng Shanthi Srinivasan