Ischemic neuronal damage specific to monkey hippocampus: Histological investigation

We previously reported lesions confined specifically to the hippocampus when produced by occluding eight vessels (the bilateral vertebral, common, internal, and external carotid arteries), which supply blood to the brain. However, histopathological changes in the primate brain, caused by ischemic injury, have not previously been thoroughly investigated. In the present study, macaque monkeys were subjected to 5–18-min ischemia by occluding the eight vessels. After the brains were perfused and fixed 5 days after the occlusion, all regions were histologically investigated for ischemic cell changes. Ischemia for 5 min produced no ischemic cell change. Ischemia for 10–15 min produced cell death limited to the deeper portion of the pyramidal cell layer of the CA1 subfield in the hippocampus. In most monkeys, no cell death was observed in any brain region outside of the hippocampus after ischemia for up to 15 min. Ischemia for 18 min produced more widespread cell death in the CA1 subfield of the hippocampus, and cell death was no longer confined to the hippocampus, but was observed in layers III, V, and VI of the neocortices, the striatum, and some other regions. Brains that were perfused and fixed 1 year after 15-min ischemic insult revealed no ischemic cell morphological change in any region, but the number of pyramidal cells in the CA1 subfield was decreased to about half. The results indicate that the CA1 subfield of the monkey hippocampus is the precise region of the brain most susceptible to ischemic insult in the primate forebrain, and after a critical time (15-min ischemia in this procedure) ischemic cell changes occur suddenly and extensively. Ischemia due to occlusion of eight arteries for 10–15 min could produce a model of human amnesia caused by transient ischemic insult.     

Calcium entry blocker ameliorates ischemic neuronal damage in monkey hippocampus.

Effects of treatment with (+/-)-1-(3,4-dimethoxyphenyl)-2-(4- diphenylmethylpiperazinyl)ethanol dihydrochloride (NC-1100), a calcium entry blocker, on ischemic neuronal damage were investigated. Monkeys were subjected to temporary occlusion of eight (bilateral common carotid, internal and external carotid, and vertebral arteries) major arteries. Blood flow was restored after 5, 10, 13, and 15 min occlusion, and NC-1100 (1 mg/kg) was then immediately infused intravenously. Monkeys were killed by perfusion fixation 5 days after occlusion. All brain regions were then histologically investigated for ischemic neuronal changes. Physiological data of NC-1100-treated subjects were not significantly different than those of untreated subjects. Heart rate tended to decrease after ischemia in treated subjects. Occlusion of 8 arteries for 10 to 15 min produced ischemic neuronal damage confined exclusively to the CA1 subfield of the hippocampus. Treatment with NC-1100 markedly reduced ischemic neuronal damage in the CA1 subfield of the hippocampus. It is suggested that postischemic treatment with the calcium entry blocker, NC-1100, might protect the brain from the ischemic damage produced in patients suffering from transient ischemia.     

Hippocampal damage following repeated brief hypotensive episodes in the rat

We examined the brain damage following repeated hypotensive episodes in the rat. Severe hypotension was induced by withdrawal of arterial blood. The MABP was maintained at about 25 mm Hg with isoelectric EEG and the shed blood was retransfused. After 1 week of recovery, histopathological changes were examined. No brain damage was observed after 1 min of isoelectric EEG. Mild neuronal damage to the hippocampal CA1 subfield was seen in some animals after two episodes of 1-min isoelectric EEG at a 1-h interval. Significant and consistent neuronal loss in the hippocampal CA1 subfield was observed after three episodes of 1-min isoelectric EEG. Scattered neuronal damage in the thalamus was additionally seen in some animals. The present study indicates that repeated brief hypotensive episodes produce brain damage depending on the number of episodes, even though no brain damage results when induced as a single insult. This animal model may reproduce hemodynamic transient ischemic attacks in humans.     

Physiological Results of Monkey Brain Ischemia, and Protection by a Calcium Blocker

Physiological and histological investigation was undertaken to examine dynamic and metabolic changes due to transient ischemic insult of the monkey brain with and without postischemic treatment by the calcium entry blocker, NC-1100 (1 mg/kg, IV). Monkeys were subjected to temporary occlusion of the eight major arteries: bilateral common carotid, internal and external carotid, and vertebral arteries. Blood flow was restored after 5-, 10-, 13-, and 15-min ischemia in different monkeys. The amplitudes of extradural, cortical, and hippocampal electroencephalograms decreased severely within 1–6 min after beginning occlusion. Complete recovery of these electroencephalograms required more than 1 h. During ischemia, significant change was obvious in arterial glucose, and systolic, diastolic, and mean blood pressure, all of which increased. There were no significant physiological differences between the untreated and NC-1100—treated groups, except decreased diastolic blood pressure and slightly lower postischemic heart rate in the treated group. These small differences might be accounted for by the effect of the calcium blocker. Ten to 15 minutes ischemia caused cell changes, including cell death, which were confined almost exclusively to the CA1 subfield of untreated hippocampi examined the fifth day after occlusion. However, no ischemia-induced cell change was observed in the CA1 subfield of hippocampi subjected to 10 to 15 min ischemia in the NC-1100-treated group. It was concluded that a calcium entry blocker can protect neurons from mild ischemia-induced injury and might ameliorate morphological damage and functional impairment of the brain due to ischemia in patients who suffer transient anoxic or hypoxic injury. The present physiological data should contribute to their clinical treatment.     

A reversible type of neuronal injury following ischemia in the gerbil hippocampus

The Mongolian gerbil is known to develop delayed neuronal death in the hippocampus following brief forebrain ischemia (Brain Res 239: 57-69, 1982). The effect of pentobarbital on this slow process of neuronal damage was examined. Immediately following 5 min of bilateral carotid occlusion, pentobarbital (10, 20, or 40 mg/kg) was injected. The control animals received saline injection. Seven days following ischemic insult, animals were perfusion-fixed and the neuronal density in the hippocampal CA1 subfield was counted. Most of the neurons in the CA1 sector survived ischemic insult when pentobarbital was given, whereas most of control group neurons were lost without the treatment. The average neuronal density of 20 mg/kg group was 168.2 +/- 12.3 (SEM) per 1 mm linear length of the CA1 subfield. The density in 40 mg/kg group was 181.1 +/- 14.9. The neuronal density in the whole control group was 34.3 +/- 5.1. The density of unoperated normal gerbils was 212.3 +/- 3.9. This result indicates that the neuronal damage of "delayed neuronal death" is reversible. On the other hand, when pentobarbital was injected 1 hr following ischemia, it showed no effect. The cell change in the CA1 sector, reversible at the initial stage, seems to rapidly become irreversible, while neurons still remain intact morphologically.     

Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat

The protective effects of protein kinase inhibitors and a calmodulin kinase inhibitor (W-7) against ischemic neuronal damage were examined in the CA1 subfield of the hippocampus. Staurosporine, KT5720, and KT5822 were used as inhibitors of protein kinase C (PKC), cyclic AMP-dependent protein kinase, and cyclic GMP-dependent protein kinase, respectively. All test compounds were injected topically into the CA1 subfield of the hippocampus. In the gerbil ischemia model, staurosporine (0.1-10 ng) administered 30 min before ischemia prevented neuronal damage in a dose-dependent manner. However, KT5720, KT5822, and W-7 were ineffective, even at a dose of 10 ng. In the rat ischemia model, staurosporine (10 ng) also prevented neuronal damage when administered before ischemic insult, although staurosporine administered 10 or 180 min after recirculation was ineffective. These results suggest the involvement of PKC in CA1 pyramidal cell death after ischemia and that the fate of vulnerable CA1 pyramidal cells through PKC-mediated processes could be determined during the early recirculation period.     

Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects

We examined the response of the gerbil brain to secondary ischemic insult following pretreatment with brief ischemia at intervals of 5 min, 1 and 6 h, 1, 2, 4, 7 and 14 days. Two minutes of bilateral carotid artery occlusion produced no histopathological brain damage, whereas 3 min of occlusion caused a moderate to severe reduction in the number of hippocampal CA1 pyramidal cells. Two-minute occlusion followed by 3-min occlusion at 5-min, 1- and 6-h intervals resulted in almost complete destruction of CA1 neurons. Additional neuronal damage was observed in the striatum at a 1-h interval and in the thalamus and the neocortex at 1- and 6-h intervals. The neuronal damage was most severe at a 1-h interval. Two-minute ischemia followed by 3-min ischemia at intervals of 1, 2, 4 and 7 days, however, caused a marked protective effect, and the hippocampal CA1 neurons were preserved. The protective effect was not observed at a 14-day interval and following pretreatment with 1-min ischemia. Thus, pretreatment with brief ischemia leads to complex responses of the brain to secondary ischemic insult; cumulative damage at intervals of 1-6 h and protective effects at intervals of 1-7 days.     

'Ischemic tolerance' phenomenon detected in various brain regions.

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the 'ischemic tolerance' phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of 'ischemic tolerance'.     

一种C-Jun N-末端激酶肽抑制剂在沙土鼠全脑缺血中的保护作用

目的 为了进一步研究海马C1区域神经细胞活动中JNK的作用，我们评价了一种JNK抑制剂即D-JNKI1在沙土鼠一过性大脑缺血模型中对迟发性神经细胞死亡（DND）的作用。方法 55只沙土鼠随机分为11个组。5组沙土鼠先接受5min前脑缺血处理，再灌注3h后，通过立体定向方法。向每组沙土鼠右侧侧脑室内分别注入不同浓度的D-JNKI1（2μL PBS内加入0．00012，0．0012，0．012，0．12，1．2μmol／L D-JNKI1，每组n=5）。对照组（n=5）：沙土鼠先接受5min前脑缺血处理，再灌注3h后，通过立体定向方法方法向右侧侧脑室内仅注入PBS2μL。腹腔内注射组（n=5）沙土鼠；先接受5min前脑缺血处理，再灌注3h后，1．2μmol／L D-JNKI1溶于0．5mL PBS腹腔内注射。假手术组（n=5）；沙土鼠仅暴露双侧颈总动脉，未夹闭。预处理组（共3组，n=15）：先将0．0012μmol／L D-JNKI1，0．00012μmol／L D-JNKI1溶于2μL PBS，分别注入两组沙土鼠的右侧侧脑室内，另外一组沙土鼠的右侧侧脑室内仅仅注入PBS2μL，30min后三组均夹闭双侧颈总动脉2min，48h后再次接受双侧颈总动脉夹闭5min。所有沙土鼠从接受夹闭5min双侧颈总动脉后4d处死，作冰冻切片和Niss1染色。结果 缺血再灌注3h后用D-JNKI-1治疗，有神经保护作用，最好的神经保护效应浓度为0．0012μmol／L。D-JNKI-1预处理加强了2min预处理所诱导的缺血耐受效应。结论D-JNKI1在沙土鼠全脑缺血模型中对海马CA1区域的迟发性神经细胞死亡有潜在的神经保护作用。     

‘Ischemic tolerance’ phenomenon detected in various brain regions

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the ‘ischemic tolerance’ phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of ‘ischemic tolerance’.     

Temporospatial expression of HSP72 and c-JUN, and DNA fragmentation in goat hippocampus after global cerebral ischemia

The role of gene induction (expression of HSP72 and c-JUN proteins) and delayed ischemic cell death (in situ labeling of DNA fragmentation) have been investigated in the goat hippocampus after transient global cerebral ischemia. The animals were subjected to 20-min ischemia (bilateral occlusion of the external carotid arteries plus bilateral jugular vein compression) and allowed to reperfuse for 2 h, and then 1, 3, and 7 days. Histological signs of cell loss were not found in the hippocampus at 2 h, 1 day, or 3 days of reperfusion. However, such an ischemic insult produced extensive, selective, and delayed degeneration in the hippocampus, as 68% of the neurons in CA1 had died at 7 days, but cell loss was not detected in CA3 and dentate gyrus fields. Concomitantly, a high percentage of TUNEL-positive CA1 neurons (60+/-9%, mean +/- SEM) was seen at 7 days, but not at the earlier time points. Mild induction of HSP72 was detected in the goat hippocampus after ischemia. The maximum percentage of HSP72-positive neurons (10-15%) was shown at 3 days of reperfusion and was concentrated mainly in the CA3 field, subiculum, and hilus, rather than in the CA1 field, whereas HSP72 expression was hardly detected at 7 days. At this later time point, scattered induction of nuclear c-JUN was found in a few neurons. The results show that: 1) postischemic delayed neuronal death selectively affects the CA1 field in the goat hippocampus, a phenomenon which seems to take longer to develop than in previously reported rodent models; and 2) postischemic expression of c-JUN does not appear to be related to cell death or survival, while the inability of most CA1 neurons to express HSP72 could contribute to neuronal death.     

Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors

This paper is to study the participation of cathepsin in ischemic neuronal death of the monkey hippocampal cornu ammonis (CA) 1 sector and also to clarify whether its selective inhibitor epoxysuccinyl peptides such as CA-074 and E-64c can inhibit the neuronal death or not. In the preceding reports, we demonstrated mu-calpain activation and subsequent rupturing of the lysosomal membrane of postischemic CA1 neurons and also increase of enzyme activity of cathepsins B and L in monkeys undergoing a complete 20-min whole brain ischemia. Here, morphological, immunohistochemical and enzymatical analyses were performed to examine the efficacy of two selective cathepsin inhibitors in the postictal blockade of delayed neuronal death in the monkey hippocampus. Both inhibitors could significantly decrease enzyme activities of cathepsins B and L in all hippocampal sectors. When CA-074 was intravenously administered immediately after the ischemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 after ischemia. In contrast, when E-64c was similarly administered, approximately 84% of CA1 neurons were saved from delayed neuronal death on day 5. The surviving neurons showed mild central chromatolysis and negligible immunoreactivity for cathepsins B and L. These observations indicate that the use of cathepsin inhibitors may become novel strategy for prevention of ischemic delayed neuronal death in the primate hippocampus.     

The neuroprotective effects of N1-dansyl-spermine in the gerbil model of cerebral ischaemia

The effects of N1-dansyl-spermine, a polyamine antagonist, and ifenprodil, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, were investigated in the gerbil model of global cerebral ischaemia. Transient forebrain ischaemia was induced by 5-min bilateral occlusion of the common carotid arteries. N1-dansyl-spermine (2, 5 and 10 mg/kg) and ifenprodil (30 mg/kg) were administered intraperitoneally 30 min after bilateral carotid artery occlusion. On histological examination, 4 days (96 h) after ischaemia, there was a significant decrease in neuronal density of the hippocampal CA1 subfield. This reduction in neuronal density was attenuated in those animals treated with the 5 or 10 mg/kg dose of N1-dansyl-spermine and those treated with 30 mg/kg ifenprodil. However, unlike ifenprodil, N1-dansyl-spermine failed to attenuate the ischaemia-induced increase in locomotor activity. This demonstrates that polyamines play a significant role in the neuronal damage produced after cerebral ischaemia, while casting doubt on the suggestion that increased locomotor activity correlates with CA1 pyramidal cell damage.     

Prevention of ischemic neuronal damage by α 1-adrenoceptor agonist (methoxamine)

The effect of methoxamine, an alpha 1-adrenoceptor agonist, on ischemic neuronal damage was studied in the gerbil. The animals were subjected to 5 min of ischemia by bilateral common carotid arteries occlusion. Morphological changes and calcium accumulation were evaluated in the CA1 sector of the hippocampus after 7 days of survival. The degree of ischemic neuronal damage and calcium accumulation in the methoxamine-treated groups were significantly attenuated compared with the saline-treated ischemic group. The results suggest that alpha 1-adrenoceptor stimulation prevents ischemic neuronal damage.     

Neuroprotective effects of creatine administration against NMDA and malonate toxicity

We have investigated the regional difference of neuronal vulnerability within the hippocampus in the C57BL/6 strain mice after forebrain ischemia. Both common carotid arteries of fifty mice were occluded for 12 min and the mouse brain was examined with cresyl violet staining. The CA4 sector was found to be the most vulnerable within the hippocampus. The CA2 and the medial CA1 sector was the 2nd and 3rd most vulnerable regions. However, The lateral part of the CA1 sector, CA3 sector and the dentate gyrus were resistant to ischemic insult.     

沙鼠脑缺血耐受的组织学变化及HSP在其中的作用

目的 :观察脑缺血耐受时的组织学变化及 HSP在其中的作用。方法 :通过 HE染色观察脑缺血耐受时的组织学变化 ,并通过免疫组化染色 ,了解 HSP70及 HSP2 7在其中的作用。结果 :一次性 5分钟缺血后 7天海马 CA1区神经元大多坏死 ,若在缺血前给予 2分钟的缺血预处理 ,该区神经元大多保留 ,表现出明显的保护作用。只给一次性 5分钟缺血 ,海马 CA1区神经元无 HSP70染色。若在缺血前给予预处理 ,海马 CA1区神经元可见明显 HSP70染色。而HSP2 7主要在胶质细胞表达 ,海马区的神经元未见其表达。结论 :缺血前给予预处理对以后的缺血有保护作用 ;在缺血耐受过程中 ,HSP70表达出一定的保护作用。     

Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil

The neuroprotective effects of MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors, were evaluated in models of cerebral ischemia using Mongolian gerbils. Bilateral occlusion of the carotid arteries for a period of 5 min resulted in a consistent pattern of degeneration of hippocampal CA1 and CA2 pyramidal neurons, which was quantified using an image analyzer. Systemic administration of MK-801 (0.01-10 mg/kg, i.p.) 1 hr prior to the occlusion caused a dose-dependent protection of the CA1 and CA2 neurons. The ED50 value for neuroprotection by MK-801 was calculated to be 0.3 mg/kg, and at doses greater than or equal to 3 mg/kg the majority of animals were completely protected against the ischemic insult. Systemic administration of MK-801 (1 or 10 mg/kg, i.p.) 1 hr prior to unilateral occlusion of the right carotid artery resulted in significant protection against hippocampal neurodegeneration following 10 min of occlusion, and increased the survival rate after 30 min of occlusion. The potent neuroprotective effects of MK-801 in these cerebral ischemia models add further weight to the evidence that NMDA receptors are involved in the mechanism of ischemia-induced neuronal degeneration.     

Naftidrofuryl protects neurons against ischemic damage

The effects of naftidrofuryl on postischemic neuronal damage and on local cerebral blood flow (LCBF) were examined in a rat model of forebrain ischemia (occlusion of carotid arteries and hypotension). Ischemia was induced for 10 min. LCBF was measured after 2 and 10 min of recirculation. A histological evaluation of cell loss in the hippocampal areas was performed 7 days after ischemia. Naftidrofuryl (10 mg/kg) was administered intraperitoneally 15 min before ischemia. The drug reduced the percentage of necrotic neurons in the CA1 and CA4 sector of the hippocampus, while the LCBF of these hippocampal sections was not significantly altered. Thus, naftidrofuryl is suggested to protect hippocampal neurons against ischemic damage mainly by a direct effect on brain parenchyma.     

Repeated unilateral carotid occlusion in Mongolian gerbils: quantitative analysis of cortical neuronal loss

Summary To develop an experimental model which enables quantitative analysis of chronic neuronal loss in the cerebral cortex, repeated ischemic insult was performed using unilateral carotid artery occulusion in Mongolian gerbils. The effect of the time interval between the repeated ischemic insult on the survival rate of the animals and the amount of cortical neuronal loss were examined. The time course of the cortical neuronal damage in repeated ischemic insult was also studied. We repeated the occlusion four times; i.e., one 10-min and three 7-min occlusion (total 31 min of ischemia). The number of animals surviving for 3 weeks after the last biochemic insult was minimum (15.4%) for animals undergoing occlusions at 1-h intervals and maximum (100%) at 24- and 48-h intervals. The number of ischemic neuronal deaths was also dependent upon the time interval, and it was so pronounced as to allow analysis at intervals of 12 hr or 24 hr in the absence of infarction in the cortex. The number of neuronal deaths could not be determined for animals with occlusion at 1-h intervals due to the production of a large infarction, with which the 3-week survival rate was minimum. The temporal profile of cortical neuronal loss in the repeated ischemic insult at 24-h intervals indicated that the number of cortical neurons significantly decreased until 7 days after the start of the ischemic procedure. This model is useful for clarifying the pathophysiology of chronically developing ischemic neuronal death.     

Enduring memory impairment in monkeys after ischemic damage to the hippocampus.

Patient RB became amnesic following an episode of global ischemia that resulted in a bilateral lesion of the CA1 field of the hippocampus. This finding suggested that damage restricted to the hippocampus is sufficient to produce clinically significant memory impairment. To evaluate further the effect of ischemic brain damage on memory, we have developed an animal model of cerebral ischemia in the monkey. Monkeys were subjected to 15 min of reversible ischemia, using a noninvasive technique involving carotid occlusion and pharmacologically induced hypotension. These monkeys sustained significant loss of pyramidal cells in the CA1 and CA2 fields of the hippocampus, as well as loss of somatostatin-immunoreactive cells in the hilar region of the dentate gyrus. Cell loss occurred bilaterally throughout the rostrocaudal extent of the hippocampus but was greater in the caudal portion. Except for patchy loss of cerebellar Purkinje cells, significant damage was not detected in areas outside the hippocampus, including adjacent cortical regions, that is, entorhinal, perirhinal, and parahippocampal cortex, and other regions that have been implicated in memory function. On behavioral tests, the ischemic monkeys exhibited significant and enduring memory impairment. On the delayed nonmatching to sample task, the ischemic monkeys were as impaired as monkeys with lesions of the hippocampal formation and adjacent parahippocampal cortex (the H+ lesion). On two other memory tasks, the ischemic monkeys were less impaired than monkeys with the H+ lesion. In neuropathological evaluations, it has always been difficult to rule out the possibility that significant areas of neuronal dysfunction have gone undetected. The finding that ischemic lesions produced overall less memory impairment than H+ lesions indicates that the ischemic monkeys (and by extension, patient RB) are unlikely to have widespread neuronal dysfunction affecting memory that was undetected by histological examination. These results provide additional evidence that the hippocampus is a focal site of pathological change in cerebral ischemia, and that damage limited to the hippocampus is sufficient to impair memory.