Impact of Single or Repeated Dose Intranasal Zinc-free Insulin in Young and Aged F344 Rats on Cognition, Signaling, and Brain Metabolism

Abstract

Novel therapies have turned to delivering compounds to the brain using nasal sprays, bypassing the blood brain barrier, and enriching treatment options for brain aging and/or Alzheimer's disease. We conducted a series of in vivo experiments to test the impact of intranasal Apidra, a zinc-free insulin formulation, on the brain of young and aged F344 rats. Both single acute and repeated daily doses were compared to test the hypothesis that insulin could improve memory recall in aged memory-deficient animals. We quantified insulin signaling in different brain regions and at different times following delivery. We measured cerebral blood flow (CBF) using MRI and also characterized several brain metabolite levels using MR spectroscopy. We show that neither acute nor chronic Apidra improved memory or recall in young or aged animals. Within 2 hours of a single dose, increased insulin signaling was seen in ventral areas of the aged brains only. Although chronic Apidra was able to offset reduced CBF with aging, it also caused significant reductions in markers of neuronal integrity. Our data suggest that this zinc-free insulin formulation may actually hasten cognitive decline with age when used chronically.

Keywords: Cognition; Diabetes; Metabolism; Vascular.

Figure 1.

Spatial learning and memory. Path length to platform across three training days is shown in (A) young and (B) aged animals receiving chronic intranasal (IN) saline, acute IN Apidra, or chronic IN Apidra. It is important to note that in the learning phase of the task, the saline groups show similar patterns as the acute insulin groups given that those animals were only exposed to insulin once, approximately 2 hours prior to the probe trial (eg, Day 4). Although all young animals learned to find the platform across days of training (two-way analysis of variance [ANOVA]; p < .0001), as a group, aged animals did not (two-way ANOVA; p = .34). This was due mostly to the poor performance seen on Day 3 in the aged animals treated chronically with IN Apidra (post hoc; p < .001). Twenty-four hours after training, probe trial data yielded changes (C) in path length in the goal quadrant during the first 30 seconds as well as (D) in the number of platform crossings. Both variables indicate poor memory recall in aged animals (path length; two-way ANOVA; p < .0001 and platform crossings; two-way ANOVA; p < .0003). Neither acute nor chronic IN insulin affected recall performance. (E) Representative paths taken by a young and an aged saline-treated animal during the first 30 seconds of the 24-hour recall trial. Data represent mean ± SEM from 6–10 animals per group.

Figure 2.

Insulin signaling. (A) The brain of each animal was cut into regions. (B) Tissue was harvested 30, 60, or 120 minutes following intranasal (IN) Apidra and was probed with pAkt and Akt antibodies. (C) Data were normalized to the saline condition on each gel and show greater increases in insulin signaling in the three ventral regions of the brain at 120 minutes post IN insulin in aged animals. (D) No increase in insulin signaling was noted in the combined dorsal regions. (E) A significant increase in the ventral regions (combined) was seen in aged compared with young animals (two-way analysis of variance [ANOVA]; p < .05) as early as 30 minutes post delivery (two-way ANOVA; p < .05). Data represent mean ± SEM from 24 animals.

Figure 3.

MRI data on cerebral blood flow (CBF). (A) Representative CBF scans (pseudocolored) from aged animals. (B) Quantification reveals a trend for a decrease in CBF with aged animals compared with young animals (two-way analysis of variance [ANOVA]; p = .07) together with a significant increase in CBF in response to chronic intranasal Apidra in aged animals only (two-way ANOVA; p < .006). Data represent mean ± SEM from 5 animals per group.

Figure 4.

Magnetic resonance spectroscopy data on several brain metabolites. (A) Representative section scan showing the region of interest around the hippocampus (left) and the resulting spectrum used to identify key metabolites (right). (B) A significant main effect of age in n-acetylaspartate (NAA) signal was noted (two-way analysis of variance [ANOVA]; p < .005) and post hoc analysis revealed a dramatic loss in signal in the aged animals treated chronically with intranasal Apidra (p > 0.01). (C) Although myo-Inositol (mI) signals were unaffected by age or treatment, (D) the ratio of NAA to mI signal was significantly reduced in aged animals chronically treated with intranasal Apidra (two-way ANOVA interaction term, p < .005). (E) A robust trend for a drug effect was also noted on the lipid peak signal at 1.3ppm (lip-13; two-way ANOVA; p = .07). Data represent mean ± SEM from 3–5 animals per group.