Diabetic autonomic neuropathy: evidence for apoptosis in situ in the rat C. GUO, A. QUOBATARI, Y. SHANGGUAN, S. HONG, J. W. WILEY & A. QUOBATARI Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA Abstract We examined the hypothesis that activation of the apoptosis cascade occurs relatively early in diabetes mellitus affecting three distinct neuronal populations that are involved in regulating gut func- tion: (i) dorsal root ganglion (DRG), (ii) vagus nodose ganglion and (iii) colon myenteric plexus.A validated streptozotocin-induced diabetic rat model and age- matched healthy controls were studied. After 4–8 weeks of diabetes the animals were anaesthetized, fixed in situ and the relevant tissues removed. After 1 month of diabetes some animals were treated with insulin for 2 weeks to restore euglycaemia. Apoptosis was measured using immunohistochemical detection of activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TU- NEL)-positive cells in adjacent sections in neurones (PGP 9.5-positive cells). The level of apoptosis was confirmed using double-label assessment of caspase-3 and TUNEL in DRG preparations.Caspase-3 immu- noreactive neurones demonstrated a range in staining intensity. When all grades of staining were included, 6–8% of the DRG, nodose ganglia and myenteric neurones were immunoreactive in the preparations from diabetic rats compared with 0.2–0.5% in con- trols. Neurones staining positive for both caspase-3 and TUNEL accounted for 1–2% of the total neuronal population in all three preparations in diabetic rats compared with 0.1–0.2% in controls (P < 0.05). Insulin treatment reversed the percentage of TUNEL-positive neurones in diabetic rats to control levels.Activation of the apoptosis cascade occurs relatively early in diabetic autonomic neuropathy and may contribute to the pathophysiology of this disorder. Keywords Diabetic neuropathy, apoptosis, autonomic neuropathy, caspases. INTRODUCTION Diabetic neuropathy is the most common peripheral neuropathy in the western hemisphere. Up to 60% of individuals with diabetes mellitus will be afflicted with neuropathy after 20 years from their diagnosis. 1 The pathophysiology of this disorder remains unre- solved but is likely multifactorial. Chronic hypergly- caemia associated with diminished insulin production or function appears to be the primary initiating factor in the pathogenesis of diabetic complications. Several hypotheses have been proposed to explain how diabetic complications occur in this milieu including increased activity of aldose reductase, impaired neurotrophin production and function, mitochondrial dysfunction associated with the formation of reactive oxygen species, altered expression/function of protein kinase C (PKC) isoforms, abnormal calcium signalling and formation of advanced glycation end products. 2–8 Diabetic autonomic neuropathy (DAN) is a well- described complication in long-term diabetes. 1 The DAN is associated with a markedly reduced quality of life and poor prognosis. The manifestations of DAN cause multiple symptoms that can involve the (i) gastrointestinal tract: oesophageal motor dysfunc- tion, diabetic gastroparesis, gall bladder atony, dia- betic enteropathy, colonic hypomotility, anorectal dysfunction; (ii) respiratory system: reduced ventila- tory drive to hypercapnia/hypoxemia, sleep apnoea; (iii) genitourinary tract: diabetic cystopathy, erectile dysfunction and (iv) cardiovascular system: resting tachycardia, reduced heart rate variability and circa- dian rhythm of heart rate and blood pressure, painless myocardial ischaemia/infarction, orthostatic hypoten- sion, exercise intolerance, perioperative instability, and sudden death. Previous studies suggest that the function of extrinsic vagal afferent and intrinsic myenteric plexus neural pathways can be impaired Address for correspondence John W. Wiley, University of Michigan Health System, General Clinical Research Center, A7007 UH, 1500 E Medical Center Dr, Ann Arbor, MI 48109-0108, USA Tel: (734) 936-8080; fax: (734) 936-4024; e-mail: jwiley@umich.edu Received: 31 January 2004 Accepted for publication: 30 March 2004 Neurogastroenterol Motil (2004) 16, 335–345 doi: 10.1111/j.1365-2982.2004.00524.x Ó 2004 Blackwell Publishing Ltd 335