Neuroprotective Efficacy and Mechanisms of Novel Pyrrolopyrimidine Lipid Peroxidation Inhibitors in the Gerbil Forebrain Ischemia Model

Abstract

A brief period of bilateral carotid occlusion (BCO)-induced forebrain ischemia in gerbils triggers neuronal degeneration and the subsequent expression of amyloid precursor protein (APP), b-amyloid protein (b-AP), and apolipoprotein E (APO-E) in the selectively vulnerable CA, region of the hippocampus. The increase in immunoreactivity is secondary to the postischemic degeneration of the CA₁ neurons and is largely astrocyte-derived as evidenced by a simultaneous increase in glial fibrillary acidic protein (GFAP) staining. Oxygen radical-induced lipid peroxidation has been strongly suggested to play a role in postischemic neuronal damage and Alzheimer's disease. Recent literature suggests a possible link between early oxidative stress and APP overexpression. Therefore, the present investigation examined the effect of two novel brain-penetrating pyrrolopyrimidine lipid peroxidation inhibitors (PNU-101033E and PNU-104067F) on CA₁ neurodegeneration and the subsequent increase in APP, b-AP, APO-E, and GFAP immunostaining at 4 days after a 5-minute episode of forebrain ischemia. Using an antibody for lipid peroxidation–derived malondialdehyde (MDA)-modified proteins, the authors also examined the effects of PNU-104067F on MDA immunostaining 2 days after ischemia, before completion of the neuronal loss. At 2 days, the authors also evaluated microglial activation using an antibody to surface major histocompatibility complex class II antigen expressed by activated microglia. Gerbils were treated at 30 mg/kg orally 30 minutes before the BCO and 2 hours after ischemia, followed by daily dosing for the next day (microglia and MDA) and the successive 3 days for APP, b-AP, APO-E, and GFAP immunostaining. APP and APO-E staining was significantly suppressed by 50% and 66%, respectively, with either compound. b-AP immunoreactivity was decreased 56% with both compounds, and GFAP expression was significantly decreased 53% (PNU-101033E) and 60.5% (PNU-104067F). There was a concomitant partial sparing of the CA₁ hippocampal neurons by both PNU-101033E and PNU-104067F (P<.01) as determined by cresyl violet histochemistry. PNU-104067F significantly inhibited lipid peroxidation-derived MDA immunostaining and microglia activation (P<.05) at 48 hours after ischemia. Brain-penetrable lipid peroxidation inhibitors may provide attenuation of various glial response proteins after ischemic injury, probably secondary to neuronal protection.

Keywords

Cerebral Ischemia Gerbil Lipid Peroxidation Pyrrolopyrimidine Microglia Astrocyte Malondialdehyde Amyloid Precursor Protein Apolipoprotein E

Amyloid precursor protein (APP) is a large transmembrane glycoprotein which is the source of the b-amyloid (b-AP), a 40–42 amino acid peptide, known to accumulate in the senile plaques and blood vessels in individuals with Alzheimer's disease (AD) (Selkoe, 1993). Deposition of b-AP after proteolytic cleavage of APP may play a key role in the pathogenesis of this disease (Hardy and Allsop, 1991; Younkin, 1991; Selkoe, 1994). Regulation of APP expression is also of interest in the context of the secondary pathophysiology of traumatic and ischemic brain injury since recent studies have revealed abnormal expression of APP in neurons and glial cells after various brain insults, including excitotoxic injury (Nakamura et al., 1992; Topper et al., 1993), mechanical trauma (Otsuka et al., 1991; Wallace et al., 1991; Sherriff et al., 1994;), and global or focal ischemia (Abe et al., 1991; Nukina et al., 1992; Stephenson et al., 1992; Wakita et al., 1992; Hall et al., 1995b). In addition, b-AP deposits have been shown to be present in 30% of fatally head-injured patients, and occur in association with increased expression of APP (Gentleman, 1993; Roberts et al., 1994). This seems consistent with the fact that prior head injury is a risk factor for development of AD (Mortimer et al., 1985; Henderson, 1988; Merz, 1989; Graves et al., 1990).

Apolipoprotein E (APO-E), is a 34-kd protein that mediates the clearance of several plasma lipoproteins (Mahley, 1988) and appears in three isoforms (E₂, E₃, E₄). There is an increased frequency of the E₄ allele in sporadic and both early and late-onset familial AD patients (Corder et al., 1993; Houlden et al., 1993; Poirier, 1993; Saunders et al, 1993). In vitro experiments indicate that the APO-E₄ form, when oxidized, binds to b-AP causing aggregation into amyloid filaments much more rapidly than the more common APO-E₃ form (Strittmatter et al., 1993a; Sanan et al., 1994). These in vitro data suggest that APO-E may play a direct role in b-AP deposition and partially explain the association of APO-E₄ and AD. Co-localization of APO-E₄ and b-AP in the plaques of AD patients support this concept (Namba et al., 1991). Using brief forebrain ischemia in gerbils, we have previously shown an increase in APP, b-AP, and APO-E immunoreactivity in the selectively vulnerable CA₁ region of the hippocampus at 4 days after the ischemic injury (Hall et al., 1995b, Ali et al., 1996). This follows the prior 84% loss of the CA₁ neurons.

In rodent forebrain ischemia models, there is a significant increase in membrane lipid peroxidation within minutes after reperfusion which continues for several days, and is believed to play a role in the delayed neuronal damage observed by 4 days after injury (Floyd and Carney, 1991, Hall et al., 1993a, Sakamoto et al., 1991). Therefore, the present investigation examined the ability of two novel brain-penetrating pyrrolopyrimidine lipid peroxidation inhibitors, PNU-101033E and PNU-104067F (Hall et al., 1995a; 1997a; Bundy et al., 1995) (Fig. 1) to decrease delayed postischemic CA₁ damage and the expression of APP, APO-E, and b-AP. We also evaluated the effects of PNU-104067F on lipid peroxidation-derived malondialdehyde (MDA), and microglial activation using immunocytochemical methods.

Fig. 1.

Chemical structures of the pyrrolopyrimidines PNU-104067F and PNU-101033E. The suffixes represent the salts of each compound: E=di-HCI; F = mono-HCI.

MATERIALS AND METHODS

Bilateral carotid occlusion global forebrain ischemia model

All experiments were performed in strict compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Pharmacia & Upjohn, Inc.

Male Mongolian gerbils (Meriones unguiculatus), obtained from Tumblebrook Farms (West Brookfield, MA) weighing 50 to 60 g were anesthetized with methoxyflurane. A 1- to 2-cm midline throat incision provided access to both carotid arteries, which were clamped with microaneurysm clips. Gerbils were placed in a warming box with an ambient temperature maintained at 37°C via a heating chamber that holds brain temperature at 35.5°C for the duration of the bilateral carotid occlusion (BCO) (Hall et al., 1993b). After 5 minutes of BCO, the clips were removed and reperfusion was allowed for either 48 or 96 hours. The animals remained in the heating chamber until they recovered from anesthesia and could regulate their own body and brain temperatures.

Tissue preparation

After either 48 or 96 hours of reperfusion (see explanations below), the animals were deeply anesthetized and perfused intracardially. Animals were perfused with cold oxygenated Krebs-Ringer bicarbonate, pH 7.2 (Sigma, St. Louis, MO) until the effluent was cleared of blood (2 minutes), followed by perfusion with cold 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 8 minutes (approximately 200 mL). Brains were removed, blocked, and post-fixed for 4 hours at 4°C. Tissues were equilibrated in increasing concentrations of sucrose in phosphate-buffered saline (PBS), pH 7.4 (10,20,30%) over a 3-day period. After complete equilibration in 30% sucrose, the brains were frozen in liquid nitrogen vapor for 10 minutes and stored at -70°C until processing. Brains were sectioned at 25 μm on a Leitz sledge microtome with a freezing stage into 0.01 mol/L PBS at 4°C (pH 7.4). The sections were mounted on gelatin-coated slides and air dried.

Immunocytochemical procedures

After rinsing in 0.01 mol/L PBS pH 7.4 (buffer), endogenous peroxidase activity was quenched by incubating the slides in 0.3% H₂O₂ in methanol for 30 minutes. After buffer rinse, the sections were blocked in normal goat serum for 20 minutes, then incubated overnight at room temperature in the primary antibody diluted in buffer. The primary antibodies were anti-APP (clone 22C11; Boehringer Mannheim), anti-b-AP (Boehringer Mannheim), anti–glial fibrillary acidic protein (GFAP) (clone G-A-5; Boehringer Mannheim), and anti-APO-E (clone 1-H4; Organon Teknika), all used at a 1:2000 dilution. The class II surface major histocompatibility complex antibody used for assessing microglial activation was MRC OX3 (code# MCA 45G; Harlan Bioproducts for Science) at 1:100. The polyclonal anti-MDA, which has been recently described (Hall et al., 1997b), was also used at a 1:100 dilution. The slides were rinsed in buffer and then incubated in secondary antibody [affinity-purified biotinylated antimouse or antirabbit immunoglobulin G (goat); ABC Elite Kit, Vector Laboratories, Inc, Burlingame, CA] according to the recommended dilutions for 2 hours. After a 10-minute buffer rinse, slides were incubated for 1 hour in the Vectastain Elite ABC Reagent according to recommended procedures. Immunocytochemical controls were also run for sham and each of the postischemic time points that were only exposed to the secondary antibody. After a 10-minute buffer rinse, the immunoreactivity was visualized by incubation for 4.5 minutes in the ABC peroxidase substrate solution with nickel chloride added for the computer-assisted densitometric analysis. Slides were rinsed in ddH₂O, dehydrated in increasing concentrations of ethanol, cleared in xylene, and mounted in DPX.

Neuronal cell counts

For the investigation of the effects of PNU-101033E and PNU-104067F on hippocampal CA₁ neuronal preservation, a 96-hour time point was selected. This was based on our previous demonstration that postischemic CA₁ degeneration with the gerbil forebrain ischemia model in our hands is complete by 72 hours (i.e., it is not greater at either 48 or 168 hours) (Hall et al., 1995b). The animals were treated with either PNU-101033E (30 mg/kg orally, n=18), PNU-104067F (30 mg/kg orally, n=20), or vehicle (40% aqueous hydroxypropyl-b-cyclodextrin; Encapsin™HPB, American Maize-Products Company, Hammond, IN, U.S.A.) (n = 20) 30 minutes before the BCO and 2 hours after the BCO, and daily thereafter for 3 days. The doses were selected based on the previous demonstration of their protective efficacy (Hall et al., 1995 a; 1997a). A group of untreated, nonischemic (sham) animals (n = 9) was also included as a control. Numbers of normal appearing medial CA₁ neurons were blindly counted in sham, vehicle, and drug-treated animals in 25-μm sections stained with cresyl violet. The number of neurons/315 mm was counted in the medial CA₁ region and averaged for the two hemispheres.

MDA and microglial immunocytochemical analysis

For the investigation of effects of PNU-104067F on MDA immunostaining and microglial activation, animals were treated with vehicle (n=8), PNU-104067F (n=8), or sham (n = 7) using the same protocol except that sacrifice occurred at 48 hours after injury. This time point was selected for two reasons. The first is that both microglial activation (Morioka et al., 1991; Gehrmann et al., 1992) and MDA immunostaining (Hall et al., 1997b) are significantly increased by 48 hours after brief forebrain ischemia, thus constituting an adequately developed end point for drug therapy. The second reason is that we wanted to investigate drug effects on these parameters at a time that preceded completion of CA₁ degeneration so that possible mechanistic conclusions could be drawn concerning the effects of pyrrolopyrimidine administration and its possible association with CA₁ preservation. The number of microglia were manually counted across the hippocampus at 200X final magnification in a 620 mm x 1240 mm area. The raw numbers were converted to the number of microglia/mm². All evaluations were blinded. Semiquantitative analysis of MDA immunostaining was made in the right medial CA₁ region of the hippocampus. MDA slides were blindly evaluated and scored from 0 to 4, based on the intensity of immunoreactivity as shown in Fig. 2 and previously described (Hall et al., 1997b).

Fig. 2.

Photomicrographic examples of the semiquantitative grading scale of the CA1 region of the hippocampus for malondialdehyde immunostaining from 0 (no staining) to 4 (most intense staining).

Quantitative computer-assisted densitometric immunocytochemical analysis of APP, APO-E, b-AP, and astrocyte activation

Immunostaining intensity for APP, APO-E, b-AP, and astrocyte activation (GFAP) in the CA₁ region of the hippocampus was measured using an Image One analysis system (Universal Imaging Corporation, Image One, West Chester, PA, U.S.A.) attached to an Olympus microscope. A 96-hour postischemic time was selected for these studies based on the prior demonstration that these parameters are significantly and maximally increased by that point (Hall et al., 1995b). The immunostained areas of the digitized CA₁ region of the hippocampus were outlined. The adjacent nonimmunostained area representing the background was outlined separately. The optical density (OD) of each of these areas was calculated by subtracting the OD of the adjacent background from the OD for the CA₁ area. For each animal, the background-corrected OD was taken for the right and left CA₁ sector and the mean used as the value for that animal. The output of the video camera was determined to be linear over the range of the OD measured in this study. A normalizing function was applied to correct for variation in intensity of illumination between sections.

Statistical analysis

All data are expressed as mean ± SD. Differences in CA₁ neuronal counts and each of the immunocytochemical end points between sham-operated (nonischemic) and postischemic animals were statistically evaluated using a one-way analysis of variance with a P<.05 required for significance.

RESULTS

Effects on 96-hour postischemic CA1 neuronal damage

Figure 3 shows that by 96 hours after ischemia, there is an 84% loss of medial CA₁ neurons in the vehicle-treated animals (sham=82.6 ± 11.8, vehicle-treated ischemic = 13.2 ± 15.5 neurons/315 μm). This is consistent with previously published reports concerning the loss of neurons in the CA₁ region after a 5-minute ischemic gerbil injury (Kirino, 1982; Hall et al., 1995a, b ). Both PNU-101033E and PNU-104067F provided significant protection against neuronal loss. The former attenuated the CA₁ damage to 44% and the latter decreased it to 60%. Figure 4 is a cresyl violet–stained photomicrograph depicting the effects of PNU-104067F on CA₁ neuronal cell loss 96 hours days after 5 minutes of forebrain ischemia.

Effects on 48-hour postischemic MDA immunoreactivity

At 48 hours after a 5-minute period of forebrain ischemia, the CA₁ neuronal loss is approximately 50% (data not shown). Figure 5 shows that by 48 hours, the mean intensity score for lipid peroxidation-related MDA immunoreactivity in vehicle-treated animals was increased more than five-fold over sham-treated animals (P<.01 versus sham). PNU-104067F significantly decreased the postischemic immunostaining by 48% (P<.05 versus vehicle) roughly proportional to the degree of CA₁ neuroprotection assessed at 96 hours.

Fig. 3.

Effects of PNU-101033E and PNU-104067F on neuronal damage in the medial hippocampal CA1 region at 96 hours after a 5-minute period of forebrain ischemia in the gerbil. Values = mean ± SD for 9 to 20 animals.

Fig. 4.

Photomicrographic examples of 96-hour postischemic necrosis in the medial hippocampal CA1 region in (A) sham-, (B) vehicle-, and (C) PNU-104067F-treated animals. Calibration bar = 250μm.

Effects on 48-hour postischemic microglial activation

Characteristic microglial activation (major histocompatibility complex II immunostaining) in the medial CA₁ region of a sham and a vehicle-treated animal is shown in Fig. 6. No activated microglia were observed in animals 48 hours after a sham-BCO. In contrast, vehicle-treated animals had 94±21 activated microglia/mm² throughout the medial CA₁ region. Treatment with PNU-104067F significantly decreased this activation by 31%, as quantified in Fig. 7.

Fig. 5.

Effect of PNU-104067F (30 mg/kg orally) on the expression of malondialdehyde immunoreactivity at 48 hours after ischemia. Values = mean ± SD for 7 to 8 animals.

Fig. 6.

Photomicrographic example of microglial activation in the medial hippocampal CA1 region of gerbils 48 hrs after a 5-minute period of forebrain ischemia in (A) sham-operated and (B) vehicle-treated animals. Calibration bar = 500 μm.

Fig. 7.

Effects of PNU-104067F (30 mg/kg orally) on microglia activation in the medial hippocampal CA1 region at 48 hours after ischemia. Values = mean ± SD for 7 to 8 animals.

Effects on postischemic APP, b-AP, APO-E, and GFAP immunoreactivity

In Fig. 8, the effects of PNU-101033E and PNU-104067F on the intensity of delayed (96-hour) postischemic CA₁ APP, b-AP, APO-E, and GFAP immunoreactivity are presented. The immunostaining for all four parameters was significantly (P<.01 versus sham) increased in vehicle-treated gerbils. However, both PNU-101033E and PNU-104067F significantly suppressed the mean increase in APP, b-AP, APO-E, and GFAP immunostaining intensity.

Fig. 8.

Effects of PNU-101033E and PNU-104067F on (A) APP, (B) b-amyloid, (C) APO-E, and (D) GFAP immunostaining in the medial hippocampal CA1 region at 96 hours after a 5-minute period of forebrain ischemia. Values = mean ± SD for 9 to 20 animals.

DISCUSSION

The present investigation has shown the ability of two novel brain-penetrating lipid-peroxidation–inhibiting pyrrolopyrimidines, PNU-101033E and PNU-104067F, to significantly attenuate delayed postischemic hippocampal CA₁ neuronal loss as well as APP, b-AP, APO-E, and GFAP immunostaining. PNU-101033E has been previously shown to decrease infarct size in mice after permanent middle cerebral artery occlusion (Hall et al., 1995a), to lessen hippocampal CA₁ necrosis in the gerbil 5-minute forebrain ischemia model (Hall et al., 1995a), and to decrease cortical necrosis in the neonatal rat hypoxic-ischemic brain injury model (Fleck and Hall, 1995). The present study confirms the ability of PNU-101033E to decrease selective vulnerability of CA₁ neurons and shows that this property is shared by the close analog PNU-104067F. In the present study, both compounds provided significant, albeit partial, protection of the selectively vulnerable hippocampal CA₁ region which is consistent with a role of oxygen radical-induced lipid peroxidation in postischemic neuronal damage (Floyd and Carney, 1991; Hall et al., 1993a; Hall, 1996). While the present mechanistically oriented study used preischemic treatment, prior work has shown that, at least for PNU-101033E, postischemic administration beginning as late as 4 hours after reperfusion still results in significant neuroprotection in the gerbil 5-minute forebrain ischemia model (Hall et al., 1995a).

In the forebrain ischemia model, there are increases in oxygen radical formation and membrane lipid peroxidation, as well as disturbances in membrane function within the first minutes after reperfusion which precede delayed CA₁ neuronal death (Floyd and Carney, 1991; Hall et al., 1993a; Kirino et al., 1993). The increased level of lipid peroxidation persists for several days after brief forebrain ischemia (Floyd and Carney, 1991). The present results show immunocytochemical evidence of CA₁ neuronal lipid peroxidative injury by 48 hours after reperfusion as judged by the significant increase in MDA-related immunostaining. The immunostaining is increased significantly when only 50% of the CA₁ neurons are lost. Because this increase occurs during the active period of degeneration, it further indicates a participatory role of lipid peroxidation in the postischemic degenerative process. PNU-104067F significantly decreased the postischemic MDA immunostaining, which is consistent with an antioxidant neuroprotective mechanism of action of the pyrrolopyrimidines.

There are several potential sources of oxygen radicals in ischemic brain (Hall, 1996). The neuronal changes that result from ischemia are accompanied by microglial activation which may contribute to postischemic free radical production (Colton and Gilbert, 1987). Microglial activation has been documented previously to be an early event in the CA₁ region of rats subjected to brief forebrain ischemia (Morioka et al., 1991; Gehrmann et al., 1992), and microglia are perhaps the initial responding glial element after neuronal injury. Consistent with their possible role in postischemic CA₁ damage is their widespread activation coincident with the time course of active CA₁ neuronal degeneration. The present study confirms the appearance of activated microglia throughout the hippocampus 48 hours after the injury which corresponds to the midpoint of neuronal cell loss. PNU-104067F was shown to suppress microglial activation, possibly contributing to the neuroprotection observed. Moreover, the fact that a lipid peroxidation inhibitor can suppress microglial activation suggests that lipid peroxidation products may play a role in triggering microglial activation although this possibility requires further study.

In addition to the potential significance of the present study to selective neuronal vulnerability in the context of brief global ischemia, the current results may also have relevance to AD. First of all, within the hippocampus, selective CA₁ degeneration, as observed after brief forebrain ischemia, is also seen in AD brains (West et al., 1994). Thus, CA₁ protective efficacy in the gerbil model may be relevant to the possibility of CA₁ neuronal preservation in AD. Second, oxygen radicals and lipid peroxidation have recently been suggested to play a role in the development of AD. Support for this view includes the findings that (1) brain reactive oxygen levels increase with age (Blass, 1993); (2) antioxidant enzymes such as superoxide dismutase are altered in AD brains (Pappolla et al., 1992); (3) there is an increase in lipid peroxidation products in the brains of AD patients (Subbarao et al., 1990; Götz et al., 1992); (4) the AD brain is more susceptible to lipid peroxidative processes (Subbarao et al., 1990; Götz et al., 1992); and (5) there is an increased level of total iron in various regions of AD brains which can drive lipid peroxidation reactions (Richardson, 1993).

Another feature of the gerbil forebrain ischemia paradigm and the present results which may be of relevance to AD concerns the demonstrated coincident increases in immunostaining for APP, b-AP, and APO-E in the selectively vulnerable CA₁ region of the hippocampus. Our previous studies have shown an increase in APP, b-AP, and APO-E immunoreactivity which is delayed until 72 hours after ischemia by which time CA₁ pyramidal neuronal degeneration is complete (Hall et al., 1995b). The timecourses of immunostaining in the current experiments were nearly identical to those in our previous work. APP has been shown to increase in response to a variety of insults, both ischemic and traumatic. APP processing occurs in one of two ways (Gandy and Greengard, 1992; Zhong et al., 1994). Normally, it is processed via the a-secretase to produce a secreted form of APP which may function to modulate cell responses to glutamate and stabilize intracellular free Ca²⁺ (Smith-Swintosky et al., 1994). Because of these potentially neuroprotective properties, the secreted form of APP may represent a trophic response after a variety of cerebral insults. However, APP can alternatively be processed to form neurotoxic amyloidogenic fragments, most notable b-AP, under conditions of neuronal degeneration (Iverfeldt et al., 1993). However, it must be cautioned that we cannot be certain that the increases in b-AP immunostaining in our experiments represent free b peptide or whether the b-AP is contained within the context of the APP molecule. Most likely some of the increase in b-AP observed from 3 to 7 days after brief forebrain ischemia represents free b-AP based on some reported differences in the overall time courses of APP versus b-AP increase (Hall et al., 1995b).

Astrocyte proliferation, as evidenced by increases in GFAP, has also been documented in the hippocampus after ischemia (Petito et al., 1990; Schmidt-Kastner et al., 1990, Hall et al., 1995b), as confirmed in the present experiments. The coincidence of astrocytic activation and the APO-E increase suggest that astrocytes are the main source of APO-E (Boyles et al., 1985; Pitas et al., 1987). Astrocytes are the main source of APO-E in the brain, and studies have shown a relationship between increased astrocytic activation and the levels of hippocampal APO-E mRNA after injury-induced degeneration (Ignatius et al., 1986; Poirier et al., 1991; Poirier et al., 1993; Ali et al., 1996). It has been suggested that APO-E may play a role in the post-traumatic neuronal repair by increasing lipid transport. Thus, the postischemic expression of APO-E, similar to that of APP discussed earlier, could represent a repair or a trophic response. Alternatively, the increased production of certain APO-E isoforms coincident with increased b-AP could conceivably be detrimental (Strittmatter et al., 1993a; 1993b; 1994). This latter possibility seems more relevant to the current results because the increase in APO-E does not occur until after CA₁ degeneration is complete.

In summary, the pyrrolopyrimidine lipid peroxidation inhibitors PNU-101033E and PNU-104067F significantly decreased both microglial activation and lipid peroxidation–related immunostaining in the hippocampal CA₁ region 48 hours before completion of CA₁ neuronal degeneration. At 96 hours, a decrease in CA₁ neuronal loss was effected by both compounds. Also at 96 hours after the ischemic insult, both PNU-101033E and PNU-104067F significantly attenuated APP, b-AP, APO-E, and GFAP immunostaining which normally increases secondarily to the degeneration of the hippocampal CA₁ neurons. The results suggest that a decrease of oxidative damage and subsequent neuronal preservation may proportionately decrease the potentially deleterious glial expression of APP, b-AP, and APO-E. Such an action may not only have an impact on delayed postischemic functional recovery but also help to retard the amyloidogenic progression of AD.

Footnotes

Acknowledgment

The authors thank Dr. Craig Thomas from Hoechst-Marion-Roussel for the supply of polyclonal antibody to malondialdehyde-modified proteins.

Abbreviations used

References

Abe

Tanzi

Kogure

(1991) Selective induction of Kunitzprotease inhibitor domain-containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex. Neurosci Lett 125:172–174

Ali

Dunn

Oostveen

Hall

Carter

(1996) Induction of apolipoprotein E mRNA in the hippocampus of the gerbil after transient global ischemia. Mol Brain Res 38:37–44

Blass

(1993) Metabolic alterations common to neural and non-neural cells in Alzheimer's disease. Hippocampus 3:45–53

Boyles

Pitas

Wilson

Mahley

Taylor

(1985) Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest 76:1501–1513

Bundy

Ayer

Banitt

Belonga

Mizsak

Palmer

Tustin

Chin

Hall

Linseman

Richards

Scherch

Sun

Yonkers

Larson

Lin

Padbury

Aaron

Mayo

(1995) Synthesis of novel 2,4 diamino-pyrrolopyrimidines with antioxidant, neuroprotective, and antiasthma activity. J Med Chem 38:4161–4163

Colton

Gilbert

(1987) Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett 223:284–288

Corder

Saunders

Strittmatter

Schmechel

Gaskell

Small

Roses

Haines

Pericak-Vance

(1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 261:921–923

Fleck

Hall

(1995) Neuroprotective effects of the 21-aminosteroid tirilazad mesylate and the pyrrolopyrimidine U-101033E following perinatal hypoxic- ischemic brain injury in the rat. J Neurotrauma 12:968

Floyd

Carney

(1991) Age influence on oxidative events during brain ischemia/reperfusion. Arch Gerontol Geriatr 12:155–177

10.

Gandy

Greengard

(1992) Amyloidogenesis in Alzheimer's disease: some possible therapeutic opportunities. Trends Pharmacol Sci 13:108–113

11.

Gehrmann

Bonnekoh

Miyazawa

Hossmann

K-A

Kreutzberg

(1992) Immunocytochemical study of an early microglial activation in ischemia. J Cereb Blood Flow Metab 12:257–269

12.

Gentleman

Lynch

Murray

Landon

Graham

Roberts

(1993) b- Amyloid protein deposition in the brain following severe head injury: a post-mortem study of 152 cases. J Neurotrauma 10(Suppl. 1):S60

13.

Götz

Freyberger

Hauer

Burger

Sofic

Gsell

Heckers

Jellinger

Hebenstreit

Frölich

Beckmann

Riederer

(1992) Susceptibility of brains from patients with Alzheimer's disease to oxygen-stimulated lipid peroxidation and differential scanning calorimetry. Dementia 3:213–222

14.

Graves

White

Koepsell

Reifler

vanBelle

Larson

Raskind

(1990) The association between head trauma and Alzheimer's disease. Am J Epidemiol 131:491–501

15.

Hall

Andrus

Althaus

VonVoigtlander

(1993a) Hydroxyl radical production and lipid peroxidation parallels selective post-ischemic vulnerability in gerbil brain. J Neurosci Res 34:107–112

16.

Hall

Andrus

Pazara

(19936) Protective efficacy of a hypothermic pharmacological agent in gerbil forebrain ischemia. Stroke 24:711–715

17.

Hall

Andrus

Smith

Fleck

Scherch

Lutzke

Sawada

Althaus

VonVoigtlander

Padbury

Larson

Palmer

Bundy

(1997a) Pyrrolopyrimidines: novel brain-penetrating antioxidants with neuroprotective activity in brain injury and ischemia models. J Pharmacol Exp Ther 281:895–904

18.

Hall

Andrus

Smith

Oostveen

Scherch

Lutzke

Raub

Sawada

Palmer

Banitt

Tustin

Belonga

Ayer

Bundy

(1995a) Neuroprotective efficacy of microvascularly-Localized versus brain-penetrating antioxidants. Acta Neurochir (Suppl) 66:107–113

19.

Hall

Oostveen

Dunn

Carter

(1995b) Increased amyloid protein precursor and apolipoprotein E immunoreactivity in the selectively vulnerable hippocampus following transient forebrain ischemia in gerbils. Exp Neurol 135:17–27

20.

Hall

(1996) Lipid peroxidation. In: Cellular and Molecular Mechamisms of Ischemic Brain Damage, Advances in Neurology, Vol 71, ( Siesjo

Wieloch

, eds), Lippincott-Raven, Philadelphia, pp 247–257

21.

Hall

Oostveen

Anderson

Thomas

(19976) Immunocytochemical method for investigating in vivo neuronal oxygen radical-induced lipid peroxidation. J Neuro Meth 76:115–122

22.

Hardy

Allsop

(1991) Amyloid deposition as the central event in the aetiology of Alzheimer's disease. Trends Pharmacol Sci 12:383–388

23.

Henderson

(1988) The risk factors for Alzheimer's disease: a review and hypothesis. Acta Psychol Scand 78:257–275

24.

Houlden

Crook

Duff

Collinge

Roques

Rossor

Hardy

(1993) Confirmation that the apolipoprotein E4 allele is associated with late onset, familial Alzheimer's disease. Neurodegeneration 2:283–286

25.

Ignatius

Gebicke-Harter

Skene

Schilling

Weisgraber

Mahley

Shooter

(1986) Expression of apolipoprotein E during nerve degeneration and regeneration. Proc Natl Acad Sci USA 83:1125–1129

26.

Iverfeldt

Walaas

Greengard

(1993) Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration. Proc Natl Acad Sci USA 90:4146–4150

27.

Kirino

(1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239:57–69

28.

Kirino

Robinson

Miwa

Tamura

Kawal

(1993) Disturbance of membrane function preceding ischemic delayed neuronal death in the gerbil hippocampus. J Cereb Blood Flow Metab 12:408–417

29.

Mahley

(1988) Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240:622–628

30.

Merz

(1989) Is boxing a risk factor for Alzheimer's disease? JAMA 261:2597–2598

31.

Mortimer

French

Hutton

Schuman

(1985) Head injury as a risk factor for Alzheimer's disease. Neurology 35:264–267

32.

Morioka

Kalehua

Streit

(1991) The microglial reaction in the rat dorsal hippocampus following transient forebrain ischemia. J Cereb Blood Flow Metab 11:966–973

33.

Nakamura

Takeda

Niigawa

Hariguchi

Nishimura

(1992) Amyloid b- protein precursor deposition in rat hippocampus lesioned by ibotenic acid injection. Neurosci Lett 136:95–98

34.

Namba

Tomonaga

Kawasaki

Otomo

Ikeda

(1991) Apolipoprotein E immunoreactivity in cerebral deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeldt-Jakob disease. Brain Res 541:163–166

35.

Nukina

Kanazawa

Mannen

Uchida

(1992) Accumulation of amyloid precursor protein and b-protein immunoreactivities in axons injured by cerebral infarct. Gerontology 38(Suppl 2):10–14

36.

Otsuka

Tomonaga

Ikeda

(1991) Rapid appearance of b-amyloid precursor protein immunoreactivity in damaged axons and reactive glial cells in rat brain following needle stab injury. Brain Res 568:335–338

37.

Pappolla

Omar

Kim

Robakis

(1992) Immunohistochemical evidence of antioxidant stress in Alzheimer's disease. Am J Path 140:621–628

38.

Petito

Morgello

Felix

Lesser

(1990) The two patterns of reactive astrocytosis in postischemic rat brain. J Cereb Blood Flow Metab 10:850–859

39.

Pitas

Boyles

Lee

Foss

Mahley

(1987) Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 917:148–161

40.

Poirier

Hess

May

Finch

(1991) Cloning of hippocampal poly(A) RNA and GFAP mRNA in hippocampus after entorhinal cortex lesioning. Mol Brain Res 11:575–580

41.

Poirier

Davignon

Boouthillier

Kogan

Gauthier

(1993) Apolipoprotein E polymorphism and Alzheimer's disease. Lancet 3442:697–699

42.

Richardson

(1993) Free radicals in the genesis of Alzheimer's disease. Ann NY Acad Sci 695:73–76

43.

Roberts

Gentleman

Lynch

Murray

Landon

Graham

(1994) b- Amyloid protein deposition in the brain after severe head injury: implications for the pathogenesis of Alzheimer's disease. J Neurol Neurosurg Psychiatry 57:419–425

44.

Sakamoto

Ohnishi

Ogawa

(1991) Relationship between free radical production and lipid peroxidation during ischemia-reperfusion injury in rat brain. Brain Res 554:186–192

45.

Sanan

Weisgraber

Russell

Mahley

Huang

Saunders

Schmechel

Wisniewski

Frangione

Roses

Strittmatter

(1994) Apolipoprotein E associates with b amyloid peptide of Alzheimer's disease to form novel monofibrils: isoform Apo E4 associates more efficiently than Apo E3. J Clin Invest 94:860–869

46.

Saunders

Strittmatter

Schmechel

(1993) Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease. Neurology 43:1467–1472

47.

Schmidt-Kastner

Freund

(1990) Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 40:599–636

48.

Selkoe

(1993) Physiological production of the b-amyloid protein and the mechanism of Alzheimer's disease. Trends Neurosci 16: 403–408

49.

Selkoe

(1994) Alzheimer's disease: a central role for amyloid. J Neuropath Exp Neurobiol 53:438–447

50.

Sherriff

Bridges

Sivalgonathan

(1994) Early detection of axonal injury after acute human head trauma using immunocyto-chemistry for beta-amyloid precursor protein. Acta Neuropathol 87:55–62

51.

Smith-Swintosky

Pettigrew

Craddock

Culwell

Rydel

Mattson

(1994) J Neurochem 63:781–784

52.

Stephenson

Rash

Clemens

(1992) Amyloid precursor protein accumulated in regions of neurodegeneration following focal cerebral ischemia in the rat. Brain Res 593:128–135

53.

Strittmatter

Weisgraber

Huang

Dong

L-M.

Salvesen

Pericak-Vance

Schmechel

Saunders

Goldgaber

Roses

(1993a) Binding of human apolipoprotein E to synthetic amyloid b-peptide. Isoform specific effects and implications for late-onset Alzheimer disease. Proc Natl Acad Sci USA 90:8098–8102

54.

Strittmatter

Saunders

Schmechel

Pericak-Vance

Enghild

Salvesen

Roses

(1993b) Apolipoprotein E: high-avidity binding to b-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer's disease. Proc Natl Acad Sci USA 90:1977–1981

55.

Strittmatter

Weisgraber

Goedert

Saunders

Huang

Corder

Dong

L-M

Jakes

Alberts

Gilbert

Han

S-H

Hulette

Einstein

Schmechell

Pericak-Vance

Roses

(1994) Hypothesis: microtubule instability and paired helical filament formation in the Alzheimer disease brain as a function of apolipoprotein E genotype. Exp Neurol 125:163–171

56.

Subbarao

Richardson

Ang

(1990) Autopsy samples of Alzheimer's cortex show increased peroxidation in vitro. J Neurochem 55:342–345

57.

Topper

Gehrmann

Banati

Schwarz

Block

Kreutzberg

(1993) Rapid appearance of amyloid precursor protein immunoreactivity following excitotoxic brain injury. J Neurotrauma 10(Suppl 1):S78

58.

Wakita

Tominoto

Akiguchi

Ohnishi

Nakamura

Kimura

(1992) Regional accumulation of amyloid B/A4 protein precursor in the gerbil brain following transient cerebral ischemia. Neurosci Lett 146:135–138

59.

Wallace

Bragin

Sambamurti

VanderPutten

Merril

Davis

Santucci

Haroutunian

(1991) Increased biosynthesis of Alzheimer amyloid precursor protein in the cerebral cortex of rats with lesions of the nucleus basalis of Meynert. Brain Res 10:173–178

60.

West

Coleman

Flood

Troncoso

(1994) Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease. Lancet 344(8925):769–772

61.

Younkin

(1991) Processing of the Alzheimer's disease Ab4 amyloid protein precursor (APP). Brain Path 1:253–262

62.

Zhong

Quon

Higgins

Higaki

Cordell

(1994) Increased amyloid production from aberrant b-amyloid precursor proteins. J Biol Chem 269:12179–12184