Amidated and Ibuprofen-Conjugated Kyotorphins Promote Neuronal Rescue and Memory Recovery in Cerebral Hypoperfusion Dementia Model

Abstract

Chronic brain ischemia is a prominent risk factor for neurological dysfunction and progression for dementias, including Alzheimer's disease (AD). In rats, permanent bilateral common carotid artery occlusion (2VO) causes a progressive neurodegeneration in the hippocampus, learning deficits and memory loss as it occurs in AD. Kyotorphin (KTP) is an endogenous antinociceptive dipeptide whose role as neuromodulator/neuroprotector has been suggested. Recently, we designed two analgesic KTP-derivatives, KTP-amide (KTP-NH2) and KTP-NH2 linked to ibuprofen (IbKTP-NH2) to improve KTP brain targeting. This study investigated the effects of KTP-derivatives on cognitive/behavioral functions (motor/spatial memory/nociception) and hippocampal pathology of female rats in chronic cerebral hypoperfusion (2VO-rat model). 2VO-animals were treated with KTP-NH2 or IbKTP-NH2 for 7 days at weeks 2 and 5 post-surgery. After behavioral testing (week 6), coronal sections of hippocampus were H&E-stained or immunolabeled for the cellular markers GFAP (astrocytes) and NFL (neurons). Our findings show that KTP-derivatives, mainly IbKTP-NH2, enhanced cognitive impairment of 2VO-animals and prevented neuronal damage in hippocampal CA1 subfield, suggesting their potential usefulness for the treatment of dementia.

Keywords: 2VO-dementia model; chronic cerebral hypoperfusion; cognitive impairment; hippocampus; kyotorphin derivatives; neuroprotection.

FIGURE 1

Time course of the experiments planned. Female Sprague–Dawley rats were divided into groups according to the type of surgery and to the injected compound during the two time periods A and B: i.e., KTP–NH₂ (32.3 mg/kg = 96 μmol/kg), IbKTP–NH₂ (24.2 mg/kg = 46 μmol/kg) or saline solution (vehicle). KTP-derivatives were administrated only to 2VO-animals. Last day of “Time period B” corresponded to the first day of behavioral testing (i.e., open-field test). Abbreviations: i.p., intra-peritoneal; 2VO, two-vessel occlusion.

FIGURE 2

Representative H&E staining photomicrographs of hippocampal sections from sham-operated and 2VO-female rats treated or not with KTP-derivatives (details in Figure 1). Histological evaluation was performed six weeks after surgery. (A): images showing ischemic lesion (white area, black arrow) in 2VO rat (12.5× magnification). Scale bar: 300 μm. (B): images showing unilateral changes of the CA1, CA2, and CA3 pyramidal cell layers in 2VO-control group (black arrows are pointing at damaged layers) (50× magnification). Scale bar: 80 μm. Abbreviations: CA, cornu ammonis; CTR, control; 2VO, two-vessel occlusion; CTR Sham, control sham-operated animals; DG, dentate gyrus.

FIGURE 3

(A) Representative confocal Z-stack images (maximum intensity) of imunostaining for GFAP (green, astrocytic marker) and NFL (red, neuronal marker) in the hippocampal CA1 subfield from sham-operated and 2VO-female rats (details in Figure 1). Nuclei is stained blue (Hoechst). Upper panels: control groups, sham and 2VO (left and right painel, respectively). Lower painels: KTP-treated 2VO groups, 2VO KTP–NH₂, and 2VO IbKTP–NH₂ (left and right painel, respectively) (400× magnification). Note a significant decrease in the NFL signal in the 2VO-control group. Scale bar: 50 μm. (B) Quantitative results of GFAP and NFL fluorescence signals. Measurements were performed bilaterally from 3 to 4 rats per group, rendering at least 6 data points per group. Abbreviations: CTR, control; 2VO, two-vessel occlusion; CTR Sham, control sham-operated animals; GFAP, glial fibrillary acidic protein; NFL, neurofilament-L protein.

FIGURE 4

Locomotion performance of KTP-treated rat females, during the 6th week after the onset of 2VO-surgery (details on chronic treatment regimen in Figure 1). Open-field test (OFT): 2VO-animals were individually placed in the center of the test apparatus 15 min after being i.p. injected with one of KTP-derivatives (KTP–NH₂ or IbKTP–NH₂), or vehicle (saline with 5% DMSO used as a control). Sham-operated animals were injected with the vehicle. Behavior was video-recorded for a 5 min time period and data are shown as % time spent resting (A), average velocity (B), % time spent in the center of the arena (C), and the number of crossings (D). In all OFT experiments, n = 5 animals per group. In (A,D): ^∗∗∗P < 0.001 vs. 2VO-control and sham-operated groups. In (B,C): ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. sham-operated group. In (A,B,D): ^##P < 0.01 vs. KTP–NH₂. Data analyzed using one way ANOVA [P = 0.0003 in (A), P = 0.0027 in (B), P = 0.0001 in (C,D)] followed by Tukey’s post test. All data are expressed as mean ± SEM. Abbreviations: CTR, control; 2VO, two-vessel occlusion; CTR sham, control sham-operated animals.

FIGURE 5

Cognitive performance in the Y-maze (A,B) and pain behavioral responses in the hot-plate (C) of KTP-treated rat females during the 6th week after the onset of 2VO-surgery (details on chronic treatment regimen in Figure 1). (A,B) Y-maze: animals were individually placed in the Start arm of the apparatus and allowed to explore freely the entire maze. Behavior was video-monitored for a 5 min time period and data are shown as number of total entries (A) and % time spent in the Other and Novel arms (B). In all Y-maze experiments, n ≥ 4 animals per group. In (B): ^#P < 0.05 vs. sham-operated group; ^∗P < 0.05, ^∗∗P < 0.01 vs. 2VO-control group, one way ANOVA [P = 0.0083 for % time in Other arm, P = 0.0283 for % time in Novel arm] followed by Tukey’s post test. Comparison between % time in Other arm and % time in Novel arm for 2VO-control group: ^§§ P = 0.0028, unpaired two-tailed Student’s t-test. (C) Hot-plate: animals were individually placed in the analgesimeter apparatus and temperature to elicit a hind paw licking or jumping recorded. In all hot-plate experiments, n ≥ 3 rats per group ^∗P = 0.0209 vs. sham-operated group; ^#P = 0.0123, ^##P = 0.0026 vs. 2VO-control group, unpaired two-tailed Student’s t-test. All data are expressed as mean ± SEM. Abbreviations: CTR, control; 2VO, two-vessel occlusion; CTR sham, control sham-operated animals.