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Central cholinergic function and metabolic changes in streptozotocin‐induced rat brain injury
(2020)
As glucose hypometabolism in the brain is an early sign of Alzheimer´s dementia (AD), the diabetogenic drug streptozotocin (STZ) has been used to induce Alzheimer‐like pathology in rat brain by intracereboventricular injection (icv‐STZ). However, many details of the pathological mechanism of STZ in this AD model remain unclear. Here, we report metabolic and cholinergic effects of icv‐STZ using microdialysis in freely moving animals. We found that icv‐STZ at a dose of 3 mg/kg (2 × 1.5 mg/kg) causes overt toxicity reflected in body weight loss. Three weeks after STZ administration, histological examination revealed a high number of glial fibrillary acidic protein reactive cells in the hippocampus, accompanied by Fluoro‐Jade C‐positive cells in the CA1 region. Glucose and lactate levels in microdialysates were unchanged, but mitochondrial respiration measured ex vivo was reduced by 9%–15%. High‐affinity choline uptake, choline acetyltransferase, and acetylcholine esterase (AChE) activities in the hippocampus were reduced by 16%, 28%, and 30%, respectively. Importantly, extracellular acetylcholine (ACh) levels in the hippocampus were unchanged and responded to behavioral and pharmacological challenges. In comparison, extracellular ACh levels and cholinergic parameters in the striatum were unchanged or slightly increased. We conclude that the icv‐STZ model poorly reflects central cholinergic dysfunction, an important characteristic of dementia. The icv‐STZ model may be more aptly described as an animal model of hippocampal gliosis.
Alzheimer’s disease (AD) is the major cause of dementia. It is characterized by the accumulation of abnormal proteins (amyloid-β plaque and neurofibrillary tangles) leading to loss of synapses, dendrites, neurons, memory and cognition. Sporadic late-onset AD is the major type of AD characterized by unclear etiology and a lack of disease-modifying therapy. To understand this disease, an alternative AD hypothesis has been proposed: AD may resemble diabetes in the brain or “diabetes type 3”. This hypothesis is supported by the fact that (1) brain glucose hypometabolism precedes AD clinical symptoms and (2) diabetes increases the risk of AD. To test this hypothesis, wild-type rats receiving intracerebroventricular administration of streptozotocin (icv-STZ) were used as a model. Streptozotocin (STZ) is a glucosamine-nitrosourea compound commonly used to induce experimental diabetes by peripheral administration. A similar pathological mechanism to peripheral STZ is then proposed to explain icv-STZ toxicity: insulin receptor signaling impairment results in glucose hypometabolism leading to cognitive deficits.
Objective: Icv-STZ model seems promising as a toxin-induced, non-transgenic AD model with the possibility to connect AD and diabetes mellitus (DM), one of the risk factors for AD. However, the mechanisms of how icv-STZ induced AD-like symptoms are unclear. Therefore, using microdialysis as the main technique, we tested 2 AD hypotheses in this model: (1) the glucose hypometabolism as an alternative AD hypothesis and (2) the cholinergic deficit as an important characteristic of AD pathology. Hippocampus was chosen because cholinergic function in this region is severely affected in AD. In comparison, the striatum was chosen because it contains cholinergic interneurons and is less affected in AD.
Methods: In this study, we used male Wistar rats of 190-220 g body weight (5 weeks of age). The rats were injected intracerebrally with STZ at a dose of 3 mg/kg (2x1.5 mg/kg; „high dose“) and 0.6 mg/kg („low dose“) with saline as control. After 21 days, samples were collected to investigate cholinergic and metabolic changes using histology, biochemistry, and neurochemistry. Brain injury was confirmed using GFAP staining and Fluoro jade staining in the hippocampus. Mitochondrial toxicity was investigated by measurement of mitochondrial
respiratory function in both hippocampus and striatum. Cholinergic markers such as acetylcholinesterase (AChE) activity, choline acetyltransferase (ChAT) activity, and choline transporter (CHT-1) activity, commonly known as high-affinity choline uptake (HACU), were measured in both hippocampus and striatum using a spectrophotometer and a scintillator.
Microdialysis is the main technique in our study. It was done in awake animals under behavioral or pharmacological stimulation. We used a self-built probe with a semi-permeable membrane (pore size of 30 kDa) that was implanted in either hippocampus or striatum. The probes were then perfused with artificial cerebrospinal fluid (aCSF) supplemented with 0.1 μM neostigmine for extracellular acetylcholine level measurement. During the perfusion, small hydrophilic compounds from brain extracellular space diffuse into the dialysates. Dialysates of 15 minutes intervals were collected for 90 minutes and used for analysis. After collection of dialysates for the first 90 minutes (basal data), rats were moved to an open field box (35x32x20 cm) for behavioral stimulation. After collection of the second 90 minute dialysates, the rats were transferred back to the microdialysis cage and dialysates were collected for another 90 minutes. On day 2, after collection of dialysates under basal conditions, 1 μM scopolamine was added to the perfusion solution for stimulation of acetylcholine release. The dialysates were also collected for 90 min followed by another 90 min of dialysis without scopolamine. The microdialysate samples were then analyzed as follows. ACh level was measured by HPLC-ECD. Glucose metabolites (glucose, lactate, pyruvate) were measured by a CMA-600 microanalyzer. An alternative energy metabolite (beta-hydroxybutyrate/BHB) was measured by GC-MS. Choline and glycerol as membrane breakdown markers were also measured by HPLC-ECD and CMA-600 microanalyzer, respectively. Markers of oxidative stress (isoprostanes) were measured using a commercially available ELISA kit.
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