The Effect of Aldehyde Dehydrogenase Activator, Alda-1®, on the Ethanol-induced Brain Damage in a Rat of Binge Ethanol Intoxication

Aims This study aimed to investigate whether an aldehyde dehydrogenase (ALDH) activator (Alda-1®) reduces neuronal damage in a rat model of binge ethanol exposure. Methods Thirty-six adolescent male rats (130-150 g) were randomly assigned into three groups: sham, ethanol-only group (25% ethanol intragastrically thrice daily for four days, approximately 10 g/kg/day) and ethanol with Alda-1® group (10 mg/kg thrice daily for four days). The ALDH activity at baseline and 90 min after the last infusion in each group was measured. Brain damage was investigated using Luxol fast blue-Cresyl violet staining in the hippocampus, CA1 and CA2/3. The activation of astrocytes and microglia was examined using immunohistochemistry for antiglial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). Results After a four-day binge, the ALDH activity level was doubled in the ethanol with Alda-1® group (mean: 7.87, SD: 0.67), whereas the levels in the sham group (mean: 4.07, SD: 0.53) and ethanol-only group (mean: 3.77, SD: 0.36) were slightly decreased. More significant neuronal shrinkage, fewer neurons, and loss of Nissl in the hippocampus were observed in the ethanol-only group compared to the ethanol with Alda-1® group. Astrocytosis and microgliosis of the hippocampus also showed increased activation in the ethanol only group compared with the ethanol with Alda-1® group. Conclusion Alda-1® administration reduces cytotoxic damage to the hippocampus in adolescent rats with binge ethanol exposure.

(iMark TM microplate absorbance reader, Bio-Rad laboratories, Inc., California, USA) was used to detect the 111 colored product with strong absorbance at 450 nm. 112 The blood ALDH activity of animals was measured before the first dose on the first day and 90 min after the 113 final dose on day four (Figure 4). Eighteen animals (6 animals of each group) were randomly selected. Tail 114 venous blood (0.3 cc) was collected and centrifuged at 3,000 RPM for 10 min.

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ALDH activity was measured according to the protocol provided by Abcam 22 .

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Behavior score

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We recorded videos to observe the animals' behavior. Videos captured for the intoxication behavior score before 119 every ethanol injection (thrice daily) ( Figure 2). Videos for the withdrawal behavior score were captured every 4 120 hours beginning 10 hours after the last infusion of ethanol for 72 hours. The rats' group types were not evident in 121 the videos. Using these videos, the blinded researcher rated the behavior (intoxication and withdrawal) scores as 122 described by previous reports 8, 23 (Figure 2).

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Tissue preparation and histopathological analysis 125 After a withdrawal period (for three days after the last dose of ethanol), animals were anesthetized with a 2:1 126 ratio of zoletil (0.7 ml/kg) and rumpun (0.3 ml/kg) and were intracardially perfused with cold saline (400 ml)

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Behavior score

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The intoxication behavioral scores (6-point scale) are presented in Figure 3, and the difference between two 168 groups is not significant (p=0.739). The average daily dose of ethanol that was determined (adjusted) by the 169 intoxication scores was 10.2±3.3 g/kg/day in group two (ethanol only) and 10±2.9 g/kg/day in group three

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The withdrawal behavioral scores are presented in Figure 4. The mean withdrawal behavioral score in the  The myelinated fibers and nerve cells were dyed blue and purple, respectively. Alda-1 ® group ( Figure 5).  The reactive expression of GFAP-a, an astrocyte marker, and Iba-1-a, a calcium binding protein expressed in  Several studies have demonstrated that the adolescent brain, especially the hippocampus, is more susceptible to alcohol-induced damage than the adult brain 32, 33 .

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To the best our knowledge, this is the first study to investigate the neuroprotective effect of the ALDH 215 activator-Alda-1 ® for binge-induced brain injury. The authors confirmed that the administration of Alda-1 ® 216 increases ALDH activity approximately twofold and reduces the cytotoxic damage to the brain, especially the 217 hippocampus in adolescent rats. In this study, the binge-induced neuronal atrophy and decreased number of

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Binge ethanol-induced thalamic injury was also demonstrated in the developing mouse brain, but it has not been 228 reported in adult models. Although chronic ethanol intoxication may induce cerebellar and thalamic damage 39 , 229 acute ethanol intoxication does not significantly impair the cerebellum and thalamus in adult rats. Thus, in this 230 study, we selected the hippocampus to show the difference in brain injury between the binge group and binge 231 with Alda-1 group.

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As previously mentioned, ethanol-induced brain damage is largely attributed to acetaldehyde accumulation.

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Aldehydes are strongly electrophilic given their terminal carbonyl groups, which makes these molecules highly Alzheimer's and prion disease. Although the mechanisms of binge-induced brain damage remain unclear, it is 252 believed that connective pathways between the frontal cortical olfactory fields and mesocorticolimbic systems 253 are highly associated with the binge-induced brain damage.  and acetaldehyde is further oxidized to acetate by ALDH2. NAD + is converted to NADH during this process. 376 Figure 6. Binge ethanol exposure induces astrocyte and microglia proliferation in the hippocampus; however, 377 these features were not distinct in the ethanol + Alda-1 ® group. Representative images of the astrocyte marker 378 GFAP (A) and the microglia marker Iba-1 (B) as well as the merged image (C).