Methohexital

Chronic brain hypoperfusion (CBH) using permanent occlusion of both common carotid arteries in an aging rat model, has been shown to mimic human mild cognitive impairment (MCI), an acknowledged high risk condition that often converts to Alzheimer's disease. An aging rat model was used to determine whether hippocampal nitric oxide (NO) is abnormally expressed following CBH for two or eight weeks. At each time point, spatial memory was measured with the Morris water maze and hippocampal A[beta] 1-40/7-42 concentrations were obtained using sandwich ELISA. Real-time amperometric measures of NO representing the constitutive isoforms of neuronal nitric oxide synthase (nNOS) and endothelial (e)NOS were also taken at each time point to ascertain whether NO levels changed as a result of CBH, and if so, whether such NO changes preceded or followed any memory or amyloid-beta pathology. We found that two weeks after CBH, NO hippocampal levels were upregulated nearly four-fold when compared to nonoccluded rats but no alteration in spatial memory of A[beta] products were observed at this time point. By contrast, NO concentration had declined to control levels by eight weeks but spatial memory was found significantly impaired and A[beta] 1-40 (but not A[beta] 1-42) had increased in the CBH group when compared to control rats. Since changes in shear stress are known to upregulate eNOS but generally not nNOS, these results suggest that shear stress induced by CBH hyperactivated vascular NO derived from eNOS in the first two weeks as a reaction by the capillary endothelium to maintain homeostasis of local cerebral blood flow. The return of vascular NO to basal levels after eight weeks of CBH may have triggered metabolic changes within hippocampal cells resulting in hippocampal dysfunction as reflected by spatial memory impairment and by accumulation of A[beta] 1-40 peptide. In conclusion, our study shows that CBH initiates spatial memory loss in aging rats thus mimicking human MCI and also increases A[beta] 1-40 in the hippocampus. The memory and amyloid changes are preceded by NO upregulation in the hippocampus. These preliminary findings may be important in understanding, at least in part, the molecular mechanisms that precede memory impairment during chronic brain ischemia and as such, the pre-clinical stage leading to Alzheimer's disease. [Neurol Res 2003; 25: 635-641]

Keywords: Nitric oxide; brain hypoperfusion; hippocampus; A[beta] peptide; spatial memory; Alzheimer's disease

INTRODUCTION

Nitric oxide (NO) is a multi-functional messenger molecule involved in vascular regulation, immune reactions and neurotransmission1. Vascular NO derived from endothelial nitric oxide synthase (eNOS) plays a major role in cerebral blood flow homeostasis and in vascular tone2.

The main source of vascular NO in mammals is derived from eNOS contained within the endothelial cells. The loss of endothelial NO impairs vascular function in part by promoting vasoconstriction, platelet aggregation, smooth muscle cell proliferation, leukocyte adhesion and greater endothelial immune cell interaction3-6. Vascular NO production from the endothelium is regulated by eNOS enzyme activity and/or NOS gene expression.

Inhibition of eNOS has been reported to reduce CBF and promote brain tissue damage after experimental focal cerebral ischemia7. It is not known what role, if any, vascular NO may play in chronic brain hypoperfusion (CBH), a blood flow deficit known to be present prior to the development of Alzheimer's disease (AD)8-11.

In the current study, we used a well-characterized aging rat model of CBH created in our laboratory12-15 that mimics mild cognitive impairment (MCI) in humans16,17. MCI has been defined as memory impairment that is abnormal for the individual's age and educational level but does not meet the criteria for dementia17. More than 50% of subjects diagnosed with MCI convert to AD within four years18.

The reason for using this rat model is that the role of CBH in producing an MCI-like state is a research topic that has received little attention. MCI-like features in our rat model are achieved when both common carotid arteries are permanently occluded for four or more weeks (Figure 1). For the first several months after CBH, our rat model shows only mild spatial memory dysfunction14,15. With time and advanced aging, CBH results in an AD-like syndrome consisting of CA1 neuronal damage, severe memory impairment, capillary distortion, oxidative stress, regional synaptic loss, neurotransmitter changes, and spreading neurodegeneration and atrophy19-23. A review of the literature reveals that carotid occlusion in humans is often associated with mild to moderate memory impairment24, a similar clinical picture to that seen in MCI.

The primary objective of this preliminary study was to test whether any NO changes in the hippocampus occur after CBH and if they do, whether they precede spatial memory impairment and/or A[beta] peptide accumulation in the hippocampal sector. A positive finding would support the notion that NO pathways may trigger memory impairment (MCI) and protein markers associated with neurodegeneration.

MATERIALS AND METHODS

Male Sprague-Dawley rats (Charles River, Montreal, Quebec, Canada) 10 months old were used. After methohexital sodium (50 mg kg^sup -1^ i.p.)-ketamine (150 mg kg^sup -1^ i.m.) anesthesia, rats were randomly separated into four groups: Groups 1 and 2 (n=13) had both common carotid arteries doubly ligated; Groups 3 and 4 (n=12) were subjected to sham occlusion. Groups 1 and 3 were kept for two weeks and groups 2 and 4 for eight weeks following CBH.

Two time points (two and eight weeks) were used following CBH in order to determine any early or late NO changes in the hippocampal brain region with respect to visuo-spatial memory impairment and A[beta] accumulation.

At two and eight weeks, rats were tested on the Morris water maze (MWM) as we have previously described8. Following MWM testing, rats were killed by decapitation and brains were rapidly removed and sagittally divided. One hemisphere was frozen in -40[degrees]C isopentane and stored in a -40[degrees]C freezer for ELISA determination of A[beta] 1-40 and A[beta] 1-42. The second hemisphere was used fresh for NO determinations.

Spatial memory testing

One of the most frequently applied relational learning tests in animals is the Morris water maze (MWM), in which the subject is required to learn complex spatial relationships based on fixed visual cues. This test has been shown to be sensitive to hippocampal function25.

Rats were tested in the MWM two and eight weeks following surgery. A working memory version of the task was done over a course of six days. The 150 cm diameter pool was divided into four quadrants (NW, SW, SE, and NE). A submerged platform (1 cm below water surface) was placed approximately 22 cm from the edge of the pool near the center of the quadrant (i.e. halfway between the two poles). The testing room was decorated with fixed visual cues placed above the pool. Each rat was given four trials per day for five days. Scores from the 20 trials/rat were summed and the mean value for each group was expressed in seconds. Each trial was started at a different pole (i.e. N, S, W, or E). The order of the poles was chosen randomly. The rat was allowed a maximum of 90 sec to find the platform. If the rat was unable to find the platform in this time, he was guided to the platform. If the rat found the platform, the time it took was recorded. The rat was allowed a 20-30 sec rest on the platform between trials and after the last trial. The platform was moved to a new quadrant every day. The quadrants were chosen in a semi-random fashion so that each quadrant was used at least once. A visible black platform was used at the end of the experiment to confirm intactness of each rat's visual ability.

Nitric oxide concentrations

For NO determination, rats were decapitated and 2 mm plugs were quickly dissected from the hippocampus. Fresh tissue plugs were placed in an incubation media and NO levels were measured amperometrically in real-time using a NO-selective probe (ISO-NOP200) and NO meter (ISO-MARK II) both manufactured by World Precision Instruments (Sarasota, FL, USA). The probe tip (200 nm diameter) was positioned 10 [mu]m above the tissue surface and constitutive NO release (nNOS, eNOS) was sensed at the nanomole range from the tissue and microvessels. NO levels were determined using a computer interfaced DUO-18 software (World Precision Instruments) and values transferred to Sigma-Plot and Sigma-Stat (Jandel, CA, USA) for graphic representation and evaluation.

ELISA determinations of A[beta] 1-40 and 1-42

Homogenization and extraction

Hippocampal tissue from rat brains were weighed and 4ml PBS + protease inhibitor cocktail (Boehringer Manneheim, Indianapolis, IN, USA) per gram weight of brain was added. Samples were homogenized using a polytron tissue homogenizer for 30 sec, and stored at -80[degrees]C until used. To prepare A[beta] extract, 200 [mu]l homogenate was mixed with 200 [mu]l of 10% SDS, 4% DEA solution and sonicated. Samples were stored overnight at 4[degrees]C, and then centrifuged at 11,000 x g for 10 min. Supernatants were collected and diluted 1 : 10 in 150mM NaCl solution, then neutralized 30 min before loading on ELISA plates by adding Triton X 100 and Tris-HCl to a final concentration of 4.5% and 100 mM respectively. The sensitivity for synthetic Ab in standard curves in SDS/DEA is about 50-100 pg ml^sup -1^ for 42 and 100 pg ml^sup -1^ for 40.

ELISA

Certified Nunc-immuno MaxiSorp ELISA plates (VWR) were coated overnight with 100[mu]l of 5 [mu]g ml^sup -1^ 4G8 antibody (Signet, Dedham, MA, USA) diluted in coating buffer (20mM NaHCO^sub 3^ pH 9.6). Wells were blocked with 0.2% BSA in PBS for 1 h at room temperature and washed with Delfia wash (Perkin Elmer, Boston, MA, USA). To make standard curves, synthetic rat A[beta] 1-40 and 1-42 (California Peptide) in 1 mg ml^sup -1^ hexafluoroisopropanol stock solutions were diluted to 1 [mu]g ml^sup -1^ in 0.5% SDS/0.2% DEA solution and left overnight at 4[degrees]C. Standard curve dilutions were prepared by diluting the 1 g ml^sup -1^ A[beta] to concentrations ranging from 2.0-0.05 ng ml^sup -1^ in 0.5% SDS, 0.2% DEA, 4.5% Triton X 100, and 100 mM Tris-HCl in 150 mM NaCl solution. 100 [mu]l of samples and standards were loaded on plates and left overnight at 4[degrees]C. Plates were washed, then 100 [mu]l of affinity purified, biotinylated R226 (A[beta] 42) or R209 (A[beta] 40) antibodies (Pankaj Mehta, Staten Island, NY, USA) diluted 1 : 1000 in 0.1% BSA in PBS were added to each well for 3 h at room temperature. After washing plates, 1 [mu]l ml^sup -1^ Streptavidin-Europium in Assay Buffer (Perkin Elmer) was added to plates for 1 h. Plates were washed and Enhancement Solution (Perkin Elmer) was added to all wells. Europium time resolved fluorescence (TRF) was measured using a Victor 2 plate reader (Perkin Elmer). Ng ml^sup -1^ A[beta] in extracts were calculated by comparing TRF values for sample dilutions to linear regions of standard curves on each plate.

Statistics

Differences among groups were analyzed statistically using Analysis of Variance (ANOVA) and the Mann-Whitney nonparametric test. A value of p

RESULTS

No sensory-motor deficits or cage behavioral changes aside from spatial memory dysfunction were observed in any rat. We have previously reported that CBH does not cause changes in mean arterial pressure, serum glucose, hematocrit or blood gases during the period of ischemia13. Post-mortem brains showed no evidence of infarction, hemorrhage or swelling in any of the four groups.

Table 1 shows a summary of our findings. The data shows that after two weeks of chronic brain hypoperfusion (CBH), NO levels in the hippocampus were 3.9 times higher in the CBH group than in nonocclusion controls. No changes in memory ability of A[beta] peptide accumulation were detected at the two-week time point between the CBH group and nonocclusion controls.

At the eight-week time point, impairment of spatial memory ability (demonstrated by an increased platform location latency in the Morris water maze) and a significant increase in A[beta] 1-40 were seen only in CBH rats. The 3.9-fold increase in NO levels observed at two weeks in CBH rats declined to the level of controls by the eighth week after CBH, indicating a disturbance of this system occurred early on (Table 1).

DISCUSSION

The findings from this study indicate that a significant upregulation of NO occurred two weeks after CBH. The levels of NO at the two-week time point were 3.9 times higher than nonoccluded controls. This increase in NO concentration declined to control levels by week 8 after CBH. Interestingly, this upregulation of NO seen two weeks after CBH preceded the spatial memory impairment measured in the Morris water maze, an impairment typically seen between two and eight weeks after CBH, depending on the rat's age and strain14,15,26. Although the NO constitutive enzymes nNOS and eNOS were not specifically measured in this study, it is believed that vascular NO derived from eNOS was responsible for the NO upregulation seen two weeks after CBH.

The reasons for this conclusion are as follows:

1. We and others have shown that following CBH in rats, cerebral blood flow (CBF) is reduced by 25%-40%13,22,23, a blood flow decline that induces direct metabolic and cellular damage over time, starting in the CA1 hippocampal region and later spreading to cortical areas19-23. CBH therefore results in early dysfunction of the hippocampal formation which manifests itself by memory deficits in animals14,15 and in humans with MCI8-11,27.

2. Other studies have reported that focal brain ischemia can inhibit eNOS activity and promote tissue damage without altering nNOS activity7. Moreover, it has been shown that brain ischemia can result in endothelial damage of rat brain28.

3. Inducible NOS (iNOS) is not detected by the amperometric technique used here since NO peaking was observed without exhibiting sustained high levels of production, thus it can not be considered to have affected NO upregulation.

4. nNOS is known to be expressed in the early phase of brain ischemia while eNOS levels generally rise in the later phase29. For example, nNOS upregulation after acute focal cerebral ischemia peaks between 24 and 48 h and disappears by day 329,30, while eNOS expression peaks after seven days and disappears by day 14(30). In our study, NO upregulation was still high at 14 days probably due to the chronic and constant nature of the hypoperfusion. However, NO concentration declined to control levels by week 8 following CBH.

Our present findings further suggest that vascular NO was upregulated during the first two weeks after CBH possibly as a response to the change in shear stress (friction of fluid flow on endothelial surface) caused by CBH31 (Figure 2). Upregulation of eNOS and activation of NO in the present study is thought to be a futile compensatory mechanism that attempts to diminish the damage posed by the persistent ischemic insult. When this attempt fails, NO returns to basal levels and may eventually undergo downregulation to suboptimal concentrations, thus creating a dyshomeostasis in blood flow regulation31. Moreover, in time, the upregulated eNOS becomes dysfunctional and produces superoxide rather than NO32. What seems clear is that NO hyperactivation can result from eNOS upregulation in response to increased shear stress32,33. Changes in shear stress are known to upregulate eNOS from mechanisms related to cerebral hypoperfusion or hyperperfusion34,35.

The role of NO in memory mechanisms has become a major research interest. Intriguing evidence reveals that endogenous NO production is important in hippocampal long term potentiation (LTP) and is needed for synaptic plasticity in the CA1(36). LTP can be blocked in a concentration-dependent manner with proteins that scavenge NO36. Brain ischemia and chronic stress conditions are intimately linked to NO pathways37,38 and often induce deficits in spatial memory by suppressing LTP39.

A less likely possibility, but one which can not be ruled out from the present data, is that NO upregulation two weeks following CBH may have resulted from abnormally activated nNOS, generating excessive concentrations of free radical NO which is toxic to surrounding neurons40. However, this condition is more often the result of sudden and total blood flow cessation, such as from ligation of the middle cerebral artery41,42 and has not been observed during CBH which is a more progressive and delayed vascular insult that terminates in neuronal damage not within days or weeks but after many months14,15.

In the hippocampus, normal nonoccluded rats have been reported to process full length APP isoforms whose synthesis increases over time after CBH43. Thus, CBH appears capable of inducing aberrant APP processing of potentially amyloidogenic A[beta] fragments in the extra-cellular compartment44. It is not clear however, whether these A[beta] peptides are reactively produced in aging rat brain as part of a neuroprotective response by injured cells or whether they are neurotoxic products formulated after CBH.

Our present findings additionally revealed that NO upregulation at two weeks after CBH preceded A[beta] peptide accumulation, specifically A[beta] 1-40 in the hippocampal region which increased significantly by eight weeks following CBH. A[beta] 1-42 remained unchanged with respect to control levels after CBH at both time points.

Since A[beta] 1-40 is reported to be associated with memory loss more than A[beta] 1-42(45), this finding is not especially surprising. However, the significance of this A[beta] 1-40 increase eight weeks after CBH remains unresolved. Is the increase due to higher expression of A[beta] 1-40 by ischemic brain cells, by a lowered degradation of this peptide's breakdown, or by some other mechanism?

It has been reported that soluble A[beta] 1-40 when injected into the CA1 subfield can produce after five days extensive neuronal degeneration through long stretches of the hippocampal region46. By contrast, soluble A[beta] 1-28 or A[beta] 1-42 showed damage only at the injection site46. Moreover, intrahippocampal injections of soluble A[beta] 1-40 but not A[beta] 1-42 can impair specific learning behavior in young rats possibly from a toxic effect on hippocampal neurons47 or by generating a pro-inflammatory reaction31,37.

Reduction of CBF has been shown to result from the intravenous administration of freshly solubilized A[beta] 1-40 in mice48,49 but not from the reverse peptide A[beta] 40-1(49). This finding implies that A[beta] 1-40 has vasoactive properties which when released, may compromise or worsen cerebral perfusion.

In AD brain, soluble A[beta] 1-40 and 1-42 levels are six times higher than control brains suggesting that these peptides are precursors of their insoluble fibrillar forms50.

These studies and our present findings suggest that soluble A[beta] 1-40 under abnormal conditions might form fibrillar aggregates in rat brain similar to the amyloid fibrils formed in AD brain. However, some factor in rat brain possibly prevents soluble A[beta] 1-42 from such fibrillar assembly51. Whether this really happens or is even necessary in explaining the 'late phase' ischemic effects of CBH remains an issue to be investigated.

What we can say, is that NO upregulation apparently precedes the memory and A[beta] 1-40 changes seen at eight weeks after CBH and that in view of this pathologic timetable, NO could be the initiator for these ischemic-related events given its documented blood flow regulatory abilities2,31. Our findings further presuppose that the cerebral endothelium, particularly involving micro-vessels in the hippocampus, could become a target for early therapy by agents that can manipulate vascular NO synthesis and release.

For example, we know CBH is the primary trigger for memory impairment in the present rat model of MCI. In other words, without CBH, there would be no memory loss in these rats. What is not known is the molecular cascade that precedes memory dysfunction which is spurred by CBH. Our study provides preliminary evidence that this molecular cascade begins with vascular NO dysfunction.

Consequently, if, as we have proposed in the past52-56, a similar cerebrovascular hypoperfusion triggers human MCI that then leads to AD (and some recent reports appear to support this suggestion8,11,27), it would be reasonable to assume that abnormal expression of NO also precedes the more downstream pathologic events taking place prior to the AD syndrome, that is, memory dysfunction and abnormal A[beta] production in the brain.

To the best of our knowledge, this is the first report showing that NO dysfunction, possibly involving vascular NO, occurs before spatial memory impairment following chronic hypopersion of the brain. A more extensive study is necessary to investigate this NO-memory loss link.

ACKNOWLEDGEMENTS

This work was supported by an Alzheimer's Association Investigator-Associated Research Grant.

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J.C. de la Torre*[dagger] B.A. Pappas[dagger], V. Prevot[double dagger], M.R. Emmerling[sec], K. Mantione[para], T. Fortin[dagger], M.D. Watson[sec] and G.B. Stefano[para]

*Department of Pathology, University of California, San Diego, CA, [dagger]Neuroscience Institute, Carleton University

[double dagger]Department of Endocrinology, University of Oregon Health Sciences, OR

[sec]Department of Neuroscience Therapeutics, Pfizer Global Research & Development, Ann Arbor, MI

[para]Neuroscience Research Institute, State University of New York, NY, USA

Correspondence and reprint requests to: J.C. de la Torre, MD, PhD, UCSD-Neuropathology, 1363 Shinly, Suite 100, Escondido, CA 92026, USA. [jdelator@nctimes.net] Accepted for publication April 2003.

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