Changes in neuromuscular structure and functions of human colon during ageing are region-dependent.

OBJECTIVE: To determine if human colonic neuromuscular functions decline with increasing age. DESIGN: Looking for non-specific changes in neuromuscular function, a standard burst of electrical field stimulation (EFS) was used to evoke neuronally mediated (cholinergic/nitrergic) contractions/relaxations in ex vivomuscle strips of human ascending and descending colon, aged 35-91 years (macroscopically normal tissue; 239 patients undergoing cancer resection). Then, to understand mechanisms of change, numbers and phenotype of myenteric neurons (30 306 neurons stained with different markers), densities of intramuscular nerve fibres (51 patients in total) and pathways involved in functional changes were systematically investigated (by immunohistochemistry and use of pharmacological tools) in elderly (≥70 years) and adult (35-60 years) groups. RESULTS: With increasing age, EFS was more likely to evoke muscle relaxation in ascending colon instead of contraction (linear regression: n=109, slope 0.49%±0.21%/year, 95% CI), generally uninfluenced by comorbidity or use of medications. Similar changes were absent in descending colon. In the elderly, overall numbers of myenteric and neuronal nitric oxide synthase-immunoreactive neurons and intramuscular nerve densities were unchanged in ascending and descending colon, compared with adults. In elderly ascending, not descending, colon numbers of cell bodies exhibiting choline acetyltransferase immunoreactivity increased compared with adults (5.0±0.6 vs 2.4±0.3 neurons/mm myenteric plexus, p=0.04). Cholinergically mediated contractions were smaller in elderly ascending colon compared with adults (2.1±0.4 and 4.1±1.1 g-tension/g-tissue during EFS; n=25/14; p=0.04); there were no changes in nitrergic function or in ability of the muscle to contract/relax. Similar changes were absent in descending colon. CONCLUSION: In ascending not descending colon, ageing impairs cholinergic function.

Gastrointestinal capacity, gut hormones and appetite change during rat pregnancy and lactation.

Pregnancy and lactation increase maternal appetite and adiposity, which in humans can lead to long-term body mass retention. Previous rat reproduction studies suggest that appetite-inhibiting gut hormone, peptide-YY (PYY), is elevated, despite hyperphagia also that gastrointestinal size increases. The present study characterised changes in orexigenic (appetite-stimulating) ghrelin and anorexigenic (appetite-inhibiting) PYY and glucagon-like peptide-1 (GLP-1), and gastrointestinal architecture during pregnancy and lactation, in matched fed and fasted plasma and gut tissue samples taken during the dark phase. Enteroendocrine cells were immunolabelled, and gut masses and lengths were measured. Fasted plasma ghrelin reduced during pregnancy: it was lowest by day 18, recovered to control values at parturition, then increased by the end of lactation. Ghrelin-immunoreactive stomach cells and stomach ghrelin concentrations were highest at birth, prior to the onset of lactation-associated hyperphagia. Plasma fed GLP-1 concentrations were elevated during pregnancy, and together with higher colon concentrations of PYY and GLP-1 during early lactation, they were associated with gastrointestinal tissue expansion, not satiety. Body mass increased during lactation, whereas white adipose tissue depots depleted. Extensive gut remodelling coincided with elevated colon concentrations of PYY and GLP-1. Modifications included stomach and caecum expansion, and duodenal, ascending and descending colon circumference increases, all peaking by day 10 of lactation; increased intestinal masses and lengths peaking at lactation day 10 for small intestine and lactation day 25 for large intestine. If these physical tissue increases persist post-partum, they could accelerate future nutrient assimilation and storage in dams, and may contribute to increased obesity risk.

Age-Associated mRNA and miRNA Expression Changes in the Blood-Brain Barrier.

Functional and structural age-associated changes in the blood-brain barrier (BBB) may affect the neurovascular unit and contribute to the onset and progression of age-associated neurodegenerative pathologies, including Alzheimer's disease. The current study interrogated the RNA profile of the BBB in an ageing human autopsy brain cohort and an ageing mouse model using combined laser capture microdissection and expression profiling. Only 12 overlapping genes were altered in the same direction in the BBB of both ageing human and mouse cohorts. These included genes with roles in regulating vascular tone, tight junction protein expression and cell adhesion, all processes prone to dysregulation with advancing age. Integrated mRNA and miRNA network and pathway enrichment analysis of the datasets identified 15 overlapping miRNAs that showed altered expression. In addition to targeting genes related to DNA binding and/or autophagy, many of the miRNAs identified play a role in age-relevant processes, including BBB dysfunction and regulating the neuroinflammatory response. Future studies have the potential to develop targeted therapeutic approaches against these candidates to prevent vascular dysfunction in the ageing brain.

Interstitial cell network volume is reduced in the terminal bowel of ageing mice.

Ageing is associated with impaired neuromuscular function of the terminal gastrointestinal (GI) tract, which can result in chronic constipation, faecal impaction and incontinence. Interstitial cells of cajal (ICC) play an important role in regulation of intestinal smooth muscle contraction. However, changes in ICC volume with age in the terminal GI tract (the anal canal including the anal sphincter region and rectum) have not been studied. Here, the distribution, morphology and network volume of ICC in the terminal GI tract of 3- to 4-month-old and 26- to 28-month-old C57BL/6 mice were investigated. ICC were identified by immunofluorescence labelling of wholemount preparations with an antibody against c-Kit. ICC network volume was measured by software-based 3D volume rendering of confocal Z stacks. A significant reduction in ICC network volume per unit volume of muscle was measured in aged animals. No age-associated change in ICC morphology was detected. The thickness of the circular muscle layer of the anal sphincter region and rectum increased with age, while that in the distal colon decreased. These results suggest that ageing is associated with a reduction in the network volume of ICC in the terminal GI tract, which may influence the normal function of these regions.

Neurogenic mechanisms in bladder and bowel ageing.

The prevalence of both urinary and faecal incontinence, and also chronic constipation, increases with ageing and these conditions have a major impact on the quality of life of the elderly. Management of bladder and bowel dysfunction in the elderly is currently far from ideal and also carries a significant financial burden. Understanding how these changes occur is thus a major priority in biogerontology. The functions of the bladder and terminal bowel are regulated by complex neuronal networks. In particular neurons of the spinal cord and peripheral ganglia play a key role in regulating micturition and defaecation reflexes as well as promoting continence. In this review we discuss the evidence for ageing-induced neuronal dysfunction that might predispose to neurogenic forms of incontinence in the elderly.

Cellular changes in the enteric nervous system during ageing.

The intrinsic neurons of the gut, enteric neurons, have an essential role in gastrointestinal functions. The enteric nervous system is plastic and continues to undergo changes throughout life, as the gut grows and responds to dietary and other environmental changes. Detailed analysis of changes in the ENS during ageing suggests that enteric neurons are more vulnerable to age-related degeneration and cell death than neurons in other parts of the nervous system, although there is considerable variation in the extent and time course of age-related enteric neuronal loss reported in different studies. Specific neuronal subpopulations, particularly cholinergic myenteric neurons, may be more vulnerable than others to age-associated loss or damage. Enteric degeneration and other age-related neuronal changes may contribute to gastrointestinal dysfunction that is common in the elderly population. Evidence suggests that caloric restriction protects against age-associated loss of enteric neurons, but recent advances in the understanding of the effects of the microbiota and the complex interactions between enteric ganglion cells, mucosal immune system and intestinal epithelium indicate that other factors may well influence ageing of enteric neurons. Much remains to be understood about the mechanisms of neuronal loss and damage in the gut, although there is evidence that reactive oxygen species, neurotrophic factor dysregulation and/or activation of a senescence associated phenotype may be involved. To date, there is no evidence for ongoing neurogenesis that might replace dying neurons in the ageing gut, although small local sites of neurogenesis would be difficult to detect. Finally, despite the considerable evidence for enteric neurodegeneration during ageing, and evidence for some physiological changes in animal models, the ageing gut appears to maintain its function remarkably well in animals that exhibit major neuronal loss, indicating that the ENS has considerable functional reserve.

Differential expression of glial cell line-derived neurotrophic factor family receptor alpha-2 isoforms in rat urinary bladder and intestine.

Neurturin (NTN) is a member of the glial cell line-derived (GDNF) family of neurotrophic factors, which act via a receptor complex composed of a signal transducing receptor, c-Ret and a glycosylphosphatidylinositol (GPI)-linked ligand binding receptor, GFRalpha. Different members of the GDNF family bind preferentially to one of four different GFRalpha receptors; NTN binds preferentially to the GFRalpha-2 receptor. Recent evidence has shown that three alternatively spliced isoforms of GFRalpha-2 occur in rodent tissues, including the rat brain, myenteric plexus and kidney, and several mouse tissues. Here we have examined the occurrence of GFRalpha-2 isoforms in the adult male rat urinary bladder by RT-PCR, in parallel with samples from the muscularis externa of the rat ileum. In contrast to the ileum, only a single GFRalpha-2 isoform, the smallest isoform, known as GFRalpha-2c, was detected in the rat urinary bladder. This differential expression of GFRalpha-2 transcripts in bladder and intestine may be related to differences in the roles of NTN in the two tissues and its actions on the neurons that innervate them.

Glucagon-like peptide-1 (GLP-1) increases in plasma and colon tissue prior to estrus and circulating levels change with increasing age in reproductively competent Wistar rats.

There is a well-documented association between cyclic changes to food intake and the changing ovarian hormone levels of the reproductive cycle in female mammals. Limited research on appetite-controlling gastrointestinal peptides has taken place in females, simply because regular reproductive changes in steroid hormones present additional experimental factors to account for. This study focussed directly on the roles that gastrointestinal-secreted peptides may have in these reported, naturally occurring, changes to food intake during the rodent estrous cycle and aimed to determine whether peripheral changes occurred in the anorexigenic (appetite-reducing) hormones peptide-YY (PYY) and glucagon-like peptide-1 (GLP-1) in female Wistar rats (32-44 weeks of age). Total forms of each peptide were measured in matched fed and fasted plasma and descending colon tissue samples for each animal during the dark (feeding) phase. PYY concentrations did not significantly change between defined cycle stages, in either plasma or tissue samples. GLP-1 concentrations in fed plasma and descending colon tissue were significantly increased during proestrus, just prior to a significant reduction in fasted stomach contents at estrus, suggesting increased satiety and reduced food intake at this stage of the cycle. Increased proestrus GLP-1 concentrations could contribute to the reported reduction in food intake during estrus and may also have biological importance in providing the optimal nutritional and metabolic environment for gametes at the potential point of conception. Additional analysis of the findings demonstrated significant interactions of ovarian cycle stage and fed/fasted status with age on GLP-1, but not PYY plasma concentrations. Slightly older females had reduced fed plasma GLP-1 suggesting that a relaxation of regulatory control of this incretin hormone may also take place with increasing age in reproductively competent females.

The TNF-α antagonist etanercept reverses age-related decreases in colonic SERT expression and faecal output in mice.

Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age-related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24-month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg-1). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to "anti-TNFα" drugs as potential treatments for age-related chronic constipation.

Plasma Ghrelin Concentrations Were Altered with Oestrous Cycle Stage and Increasing Age in Reproductively Competent Wistar Females.

Changes in appetite occur during the ovarian cycle in female mammals. Research on appetite-regulatory gastrointestinal peptides in females is limited, because reproductive changes in steroid hormones present additional experimental factors to control for. This study aimed to explore possible changes in the orexigenic (appetite-stimulating) gastrointestinal peptide hormone ghrelin during the rodent oestrous cycle. Fed and fasted plasma and stomach tissue samples were taken from female Wistar rats (32-44 weeks of age) at each stage of the oestrous cycle for total ghrelin quantification using radioimmunoassay. Sampling occurred during the dark phase when most eating takes place in rats. Statistical analysis was by paired-samples t-test, one-way ANOVA on normally distributed data, with Tukey post-hoc tests, or Kruskal-Wallis if not. GLM univariate analysis was used to assess main effects and interactions in ghrelin concentrations in the fed or fasted state and during different stages of the ovarian cycle, with age as a covariate. No consistent fed to fasted ghrelin increases were measured in matched plasma samples from the same animals, contrary to expectations. Total ghrelin concentrations did not significantly change between cycle stages with ANOVA, in either fed or fasted plasma or in stomach tissue. This was despite significantly decreased fasted stomach contents at oestrus (P = 0.028), suggesting decreased food intake. There was however a significant interaction in ghrelin plasma concentrations between fed and fasted proestrus rats and a direct effect of age with rats over 37 weeks old having lower circulating concentrations of ghrelin in both fed and fasted states. The biological implications of altered ghrelin plasma concentrations from 37 weeks of age are as yet unknown, but warrant further investigation. Exploring peripheral ghrelin regulatory factor changes with increasing age in reproductively competent females may bring to light potential effects on offspring development and nutritional metabolic programming.

Ageing of the enteric nervous system.

The intrinsic neurones of the enteric nervous system (ENS) play a fundamental role in the regulation of gastrointestinal functions. Although much remains to be learnt about the changes that take place in intestinal nerves during ageing, evidence suggests that selective neurodegeneration may occur in the ageing ENS. Age-associated changes in intestinal innervation may contribute to the gastrointestinal disorders that increase in incidence in the elderly, such as dysphagia, gastrointestinal reflux and constipation. A number of other factors, such as immobility, co-morbidity, and side effects of therapeutic medication for other disorders however, are also likely to contribute to the aetiology of these conditions. An important finding in rodents is that the neuronal losses that take place in the ENS during ageing may be prevented by calorie restriction; an indication that diet may influence gastrointestinal ageing. Thus, it is of importance to understand not only how the ENS changes during 'normal' ageing, but also how external factors contribute to these changes. Here, current knowledge of how intestinal innervation is affected during normal ageing and how these changes may impact upon gastrointestinal physiology are reviewed.

Identification of GFR alpha-2 isoforms in myenteric plexus of postnatal and adult rat intestine.

Glial cell line-derived neurotrophic factor family receptor alpha-2 (GFR alpha-2) is a GPI-linked receptor that preferentially binds neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family. Three splice isoforms of GFR alpha-2 have been identified previously in mouse tissues, but the occurrence of splice isoforms in rats has not been described. The aim of this study was therefore to identify GFR alpha-2 splice isoforms in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and gene cloning. Three isoforms were identified and sequenced, and named GFR alpha-2(a), (b) and (c), according to the nomenclature used for the previously identified mouse isoforms. The GFR alpha-2(a) and (b) isoforms were identical to those previously described in mice. The GFR alpha-2(c) isoform was novel. Sequences for GFR alpha-2(b) and (c) were deposited in the GenBank database (accession numbers GI: 16797788 and 16797786, respectively). All three isoforms were expressed in the brain, kidney, and intestine of both postnatal and adult rats.

Aging of the mammalian gastrointestinal tract: a complex organ system.

Gastrointestinal disorders are a major cause of morbidity in the elderly population. The gastrointestinal tract is the most complex organ system; its diverse cells perform a range of functions essential to life, not only secretion, digestion, absorption and excretion, but also, very importantly, defence. The gastrointestinal tract acts not only as a barrier to harmful materials and pathogens but also contains the vast number of beneficial bacterial populations that make up the microbiota. Communication between the cells of the gastrointestinal tract and the central nervous and endocrine systems modifies behaviour; the organisms of the microbiota also contribute to this brain-gut-enteric microbiota axis. Age-related physiological changes in the gut are not only common, but also variable, and likely to be influenced by external factors as well as intrinsic aging of the cells involved. The cellular and molecular changes exhibited by the aging gut cells also vary. Aging intestinal smooth muscle cells exhibit a number of changes in the signalling pathways that regulate contraction. There is some evidence for age-associated degeneration of neurons and glia of the enteric nervous system, although enteric neuronal losses are likely not to be nearly as extensive as previously believed. Aging enteric neurons have been shown to exhibit a senescence-associated phenotype. Epithelial stem cells exhibit increased mitochondrial mutation in aging that affects their progeny in the mucosal epithelium. Changes to the microbiota and intestinal immune system during aging are likely to contribute to wider aging of the organism and are increasingly important areas of analysis. How changes of the different cell types of the gut during aging affect the numerous cellular interactions that are essential for normal gut functions will be important areas for future aging research.

Impaired colonic motility and reduction in tachykinin signalling in the aged mouse.

Ageing is associated with an increased incidence of constipation in humans. The contribution that the ageing process makes to this condition is unclear. The aim of this study was to determine the effects of age on faecal output and colonic motility in male C57BL/6J mice and to determine the role that altered tachykinin signalling plays in this process. Total faecal output recorded over a 24h period decreased with age due to a reduction in the number of pellets produced and their water content. These changes occurred in the absence of any significant change in food and water intake. There was an increase in the amount of faecal matter stored in the isolated colon with age which caused a proportional increase in colonic length. Analysis of colonic motility using an artificial pellet demonstrated that pellets moved in a stepwise fashion through the colon. There was an age-related increase in pellet transit time due to decreases in the step distance, velocity, and frequency of stepwise movements. These changes were reversed using the neurokinin 2 (NK2) receptor agonist neurokinin A. Addition of the NK2receptor antagonist GR159897 significantly increased transit time in the young animals by decreasing step distance, velocity and frequency, but was without effect in the aged colon. In summary, the ageing C57BL/6J mouse shows an impaired motility phenotype. These effects appear, at least in part, to be due to an attenuation of tachykinin signalling via NK2 receptors.

Loss of UCHL1 promotes age-related degenerative changes in the enteric nervous system.

UCHL1 (ubiquitin carboxyterminal hydrolase 1) is a deubiquitinating enzyme that is particularly abundant in neurons. From studies of a spontaneous mutation arising in a mouse line it is clear that loss of function of UCHL1 generates profound degenerative changes in the central nervous system, and it is likely that a proteolytic deficit contributes to the pathology. Here these effects were found to be recapitulated in mice in which the Uchl1 gene had been inactivated by homologous recombination. In addition to the previously documented neuropathology associated with loss of UCHL1 function, axonal swellings were detected in the striatum. In agreement with previously reported findings the loss of UCHL1 function was accompanied by perturbations in ubiquitin pools, but glutathione levels were also significantly depleted in the brains of the knockout mice, suggesting that oxidative defense mechanisms may be doubly compromised. To determine if, in addition to its role in the central nervous system, UCHL1 function is also required for homeostasis of the enteric nervous system the gastrointestinal tract was analyzed in UCHL1 knockout mice. The mice displayed functional changes and morphological changes in gut neurons that preceded degenerative changes in the brain. The changes were qualitatively and quantitatively similar to those observed in wild type mice of much greater age, and strongly resemble changes reported for elderly humans. UCHL1 knockout mice should therefore serve as a useful model of gut aging.

Differing effects of NT-3 and GDNF on dissociated enteric ganglion cells exposed to hydrogen peroxide in vitro.

Oxidative stress is widely recognized to contribute to neuronal death during various pathological conditions and ageing. In the enteric nervous system (ENS), reactive oxygen species have been implicated in the mechanism of age-associated neuronal loss. The neurotrophic factors, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF), are important in the development of enteric neurons and continue to be expressed in the gut throughout life. It has therefore been suggested that they may have a neuroprotective role in the ENS. We investigated the potential of NT-3 and GDNF to prevent the death of enteric ganglion cells in dissociated cell culture after exposure to hydrogen peroxide (H(2)O(2)). H(2)O(2) treatment resulted in a dose-dependent death of enteric neurons and glial cells, as demonstrated by MTS assay, bis-benzimide and propidium iodide staining and immunolabelling. Cultures treated with NT-3 prior to exposure showed reduced cell death compared to untreated control or GDNF-treated cultures. GDNF treatment did not affect neuronal survival in H(2)O(2)-treated cultures. These results suggest that NT-3 is able to enhance the survival of enteric ganglion cells exposed to oxidative stress.

Ageing of enteric neurons: oxidative stress, neurotrophic factors and antioxidant enzymes.

BACKGROUND: Ageing is associated with gastrointestinal dysfunction, which can have a major impact on quality of life of the elderly. A number of changes in the innervation of the gut during ageing have been reported, including neuronal loss and degenerative changes. Evidence indicates that reactive oxygen species (ROS) are elevated in ageing enteric neurons, but that neurotrophic factors may reduce generation of neuronal ROS. Two such factors, glial cell line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) have also been found to protect enteric neurons against oxidative stress induced cell death of enteric ganglion cells in vitro. We have investigated the possible roles of neurotrophic factors further, by examining their expression in the gut during ageing, and by analysing their effects on antioxidant enzyme production in cultures of enteric ganglion cells. RESULTS: Analysis of the expression of GDNF and its receptors c-Ret and GFR α - 1 in rat gut by RT-PCR showed that expression continues throughout life and into ageing, in both ad libitum(AL) and calorically-restricted (CR) animals. Levels of expression of GDNF and GFR α - 1 were elevated in 24 month AL animals compared to 24 month CR animals, and to 24 CR and 6 month control animals respectively. The related factor Neurturin and its receptor GFR α - 2 were also expressed throughout life, the levels of the GFR - α-2(b) isoform were reduced in 24 m AL animals. Immunolabelling showed that c-Ret and GFR α - 1 proteins were expressed by myenteric neurons in ageing animals. GDNF, but not NT-3, was found to increase expression of Cu/Zn superoxide dismutase and catalase by cultured enteric ganglion cells. CONCLUSIONS: The neurotrophic factors GDNF and neurturin and their receptors continue to be expressed in the ageing gut. Changes in the levels of expression of GDNF , GFR α-1 and GFR α-2(b) isoform occurred in 24 m AL animals. GDNF, but not NT-3, increased the levels of antioxidant enzymes in cultured enteric ganglion cells, indicating a possible mechanism for the reported protective effect of GDNF against menadione-induced neuronal apoptosis in the ageing gut. Together these data suggest that GDNF family members may play a protective role in the gut throughout life, and support the suggestion that dysregulation of neurotrophic factor support could contribute to neuronal ageing in the gut.

Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response.

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro-inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence-like state in mature postmitotic neurons in vivo. About 40-80% of Purkinje neurons and 20-40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL-6 production, heterochromatinization and senescence-associated ß-galactosidase activity. Frequencies of these senescence-like neurons increased with age. Short-term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late-generation TERC-/- mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late-generation TERC-/-CDKN1A-/- mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence-like phenotype in neurons, as in senescing fibroblasts and other proliferation-competent cells. We conclude that a senescence-like phenotype is possibly not restricted to proliferation-competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence-like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.

Colostrinin alleviates amyloid-beta induced toxicity in rat primary hippocampal cultures.

Colostrinin (CLN), a complex mixture of proline-rich polypeptides derived from colostrums, can alleviate cognitive decline in early Alzheimer's disease patients. The molecular basis of the action of CLN has been studied in vitro using human neuroblastoma cell lines. The aim of the present study was to use quantitative immunocytochemistry and immunoblotting to investigate the ability of CLN to relieve amyloid-beta (Abeta)-induced cytotoxicity in rat primary hippocampal neuronal cells. Our data confirm that CLN alleviates the effect of Abeta-induced cytotoxicity and causes a significant reduction in the elevated levels of the antioxidant enzyme SOD1.