Neuroscience Research 63 (2009) 129–137

Amyloid-b25–35 impairs memory and increases NO in the temporal cortex of rats

I. Daniel Limon a,b,d, Alfonso Dıaz a,d, Liliana Mendieta a, German Chamorro b,Blanca Espinosa c, Edgar Zenteno e, Jorge Guevara e,*a Laboratorio de Neurofarmacologıa, Facultad de Ciencias Quımicas, Benemerita Universidad Autonoma de Puebla, Puebla, Mexicob Laboratorio de Toxicologıa Preclınica, Escuela Nacional de Ciencias Biologicas IPN, D.F., Mexicoc Departamento de Bioquımica, Instituto Nacional de Enfermedades Respiratorias, D.F., Mexicod Laboratorio de Enfermedades Neurodegenerativas Instituto Nacional de Neurologıa MVS, D.F., Mexicoe Departamento de Bioquımica, Facultad de Medicina, UNAM, D.F., Mexico

A R T I C L E I N F O

Article history:

Received 10 October 2008

Received in revised form 5 November 2008

Accepted 10 November 2008

Available online 25 November 2008

Keywords:

Memory

b-Amyloid

Oxidative stress

Neurotoxicity

Nitric oxide

Nitric oxide synthase

Temporal cortex

Alzheimer’s disease

A B S T R A C T

b-Amyloid plays an important role in the neurodegeneration process of Alzheimer’s disease (AD), but its

neurotoxic mechanisms are not clear. It has been associated with the increase of oxidative stress and

cognitive impairment because the b-amyloid peptide 25–35 (Ab(25–35)) has the critical neurotoxic

properties of the full-length Ab1–42. Our present study shows the role of Ab(25–35) when injected into the

temporal cortex on the nitric oxide pathways, 3-nitrotyrosine, neuronal death, and the spatial memory of

rats 1 month after the injection. Our data showed that Ab(25–35) increases oxidative stress, causes

neuronal damage, and decreases spatial memory in rats. Notably, the injection of the fraction Ab(25–35)

caused an increase of nNOS and iNOS immunoreactivity in the temporal cortex and hippocampus. We

demonstrated a significant increase of reactive astrocytosis, which was accompanied by neuronal

damage in the temporal cortex and hippocampus of rats injected with Ab(25–35). These data suggest that

the fraction Ab(25–35) injected into the temporal cortex might contribute to understanding the role of

nitric oxide on the biological changes related to the neuropathological progression and the memory

impairment in AD.

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1. Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder.Currently it is known that brains of AD patients are characterizedby extracellular plaques of b-amyloid (Ab), neurofibrillary tanglesof tau protein, and loss of memory (Selkoe, 1996). Extensiveresearch indicates that Ab plays a pivotal role in this neurode-generation process. Amyloid plaques appear in the early stages ofAD in the temporal cortex (TCx), entorhinal cortex, and thehippocampus (Hp) (Braak and Braak, 1991; Duyckaerts, 2004; Thalet al., 2000, 2002). It is the peptide that appears to be responsiblefor cognitive impairment and degeneration in AD (Holscher et al.,2006; Yankner, 1996; Maurice et al., 1996; Cutler et al., 2004;Riepe, 2005).

Recent research in models in vivo and in vitro shows that thepeptide Ab(25–35) has the critical neurotoxic properties of the full-length Ab(1–42) (Butterfield and Boyd-Kimball, 2005). Acute

* Corresponding author at: Departamento de Bioquımica, Edificio ‘‘D’’, Facultad

de Medicina, Ciudad Universitaria, Universidad Nacional Autonoma de Mexico

(UNAM), Col. Copilco, Del Coyoacan, 04510 Mexico, D.F., Mexico.

E-mail address: Guevara.Jorge03@gmail.com (J. Guevara).

0168-0102/$ – see front matter � 2008 Elsevier Ireland Ltd and the Japan Neuroscienc

doi:10.1016/j.neures.2008.11.006

intracerebroventricular (icv) injection of Ab(25–35) is involved inmemory impairment (Pike et al., 1995; Delobbette et al., 1997;Stepanichev et al., 2000, 2003, 2006; Cheng et al., 2006).Investigations with the use of Ab(25–35) in animal models haveallowed understanding of its implications in AD-related mechan-isms. We have also demonstrated that Ab(25–35) injected into thehippocampus of rats causes a detriment to the spatial memory andenhances oxidative stress (Perez-Severiano et al., 2004). Theincrease of Ab production and its neurotoxic properties suggestthat amyloid aggregation is the primary pathogenic mechanism inAD. However, the precise mechanism by which Ab causes neuronaldeath and loss of memory remains unclear (Turner et al., 2003).

Most evidence points to the hypothesis that Ab may trigger abiochemical response from the brain, including a greater produc-tion of nitric oxide (NO). NO is produced by the three enzymes,neuronal nitric oxide synthase (nNOS), inducible nitric oxidesynthase (iNOS), and endothelial nitric oxide synthase (eNOS). Innormal conditions NO appears to participate in different processes,such as the regulation of the cerebral vasoactivity, learning andmemory, and synaptogenesis (Law et al., 2001). In recent years,high amounts of NO have been suggested the existence of apossible association between NO neurotoxicity and AD, althoughthe nNOS and iNOS activity appears to be involved in NO

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I.D. Limon et al. / Neuroscience Research 63 (2009) 129–137130

neuroprotective effects or mediated neurotoxicity in the brain(Norris et al., 1996).

Furthermore, the effect of Ab toxicity may affect NOS-containing cortical neurons (Hu and el-Fakahany, 1993). Abactivates glial cells that cause an increase in NO production, tumornecrosis factor-alpha (TNF-alpha), and proinflammatory cytokines.The interaction of NO and the superoxide anion (O2

�) also resultsin peroxynitrite and then nitration of proteins that leads to cellulardamage (Koppal et al., 1999; Meda et al., 2001; Lee et al., 2002; Tanet al., 1999; Masliah et al., 1998; Stepanichev et al., 2006). Itappears that Ab causes the increase of intracellular calcium levelsthat result in overproduction of NO, membrane lipid peroxidation,and neuronal death (Mattson et al., 1997; Mattson and Chan, 2003;Mattson, 2004). Other studies in the mouse or rat model of AD havefound the induction of oxidative stress and an increase of proteinnitration and 4-hydroxy-2-nonenal (4-HNE) as protein andlipoperoxidation markers associated with fibrillar Ab deposits(Matsuoka et al., 2001).

Our aim was to investigate the role of Ab(25–35) injected into thetemporal cortex of rats by evaluating nitric oxide production, NOSactivity, 3-nitrotyrosine (3-NT), and the spatial memory of rats.

2. Materials and methods

2.1. Animals

Adult male Wistar rats (230–250 g, n = 26) were obtained fromBioterio Claude Bernard BUAP, our animal facilities. Animals wereindividually housed in a temperature and humidity-controlledenvironment in a 12 h:12 h light–dark cycle with free access tofood and water. All the procedures described in this study are inaccordance with the Guide for the Care and Use of LaboratoryAnimals of the Mexican Council for Animal Care.

2.2. Preparation of aggregated Ab(25–35) and surgery

Aggregated, Ab(25–35) (Sigma–Aldrich Ltd., St. Louis, MO, USA)was solubilized in sterile isotonic saline solution, pH 7.4, to a finalconcentration of 100 mM. The Ab(25–35) solution was thenincubated at 37 8C for 36 h.

All animals were anesthetized with chloral hydrate (300 mg/kg,i.p.). The rats were randomly divided into two groups (n = 13) forthe stereotaxic surgery (Stoelting, IL, USA). One group was assignedto the bilateral injection of 1-mL isotonic saline solution (SSI orvehicle). A second group was used for the injection of 1-mL 100 mMAb(25–35) into the temporal cortex (coordinates: A: �4.8 mm frombregma, L: �6.2 mm from midline, V: �5.5 below dura in accordancewith Paxinos and Watson (1998).

2.3. Morris water-maze spatial-reference task

The Morris water maze (MWM) was run as described (Morris,1984). The pool was located in a large test room, in which therewere several external cues to the maze. The apparatus consisted ofa circular water pool (140-cm diameter and 80-cm high). Aplatform (20-cm diameter and 40-cm high) was set inside the pool,which was filled with water to a height of 42 cm at 23 � 2 8C. Thepool was conceptually divided into four quadrants and had fourpoints designed as starting positions (N, S, E, or W). The platform waslocated in a constant position in the middle of one quadrantequidistant from the center and the edge of the pool and it wassubmerged 2 cm below the water surface and the water was dyedwith 0.01% white titanium oxide (TiO2). In every trial, the rat wasplaced in the water facing the wall of the tank, in one of the fourpossible starting locations. The order of starting points was variedeveryday and any given sequence was not repeated on acquisition

phase days. Rats received 4 trials/day, with a 50-min intertrialinterval. This procedure was repeated for 5 training days. The rat wasallowed to search for the platform for 90 s, and was gently guided to itif it did not reach the target. If the rat found the platform, it wasallowed to remain there for 15 s and was then returned to its homecage. If the rat were unable to find the platform within 90 s thetraining session was finished and the maximum score of 90 s wasassigned.

The memory test was done 10 days after the training; allanimals received only one trial, as above described, with theplatform removed. Trials were recorded by a video cameramounted above the center of the pool (Sharp VL-WD450 U).

Three variables were measured the time to find platform (or thetime to cross over the platform location); the number of crossingson the platform quadrant (or the quadrant which the platform waslocated in training sessions) and the number of crossings on theopposite quadrants.

2.4. Nitrite assay

Animals were decapitated after the memory test (n = 7) andtheir brains were immediately removed, washed in ice-cold SSI,and the hippocampus and TCx were dissected. These werehomogenized in 3-mL ice-cold 0.1 M phosphate-buffered saline(PBS), and pH 7.4. The homogenate was centrifuged at 12,500 rpm(4 8C). The supernatant was obtained and stored at �70 8C.Supernatants were used for protein and nitrite level measure-ments. Nitrite levels were measured with the Griess method(Green et al., 1982). The Griess reagents were sulfanilamide, glacialacetic acid, N-(1-napthyl) ethylenediamine, and sodium nitrite(Sigma, St. Louis, MO, USA). The reagent, 200 mL was added directlyto an aliquot of the homogenate (200 mL) and incubated underreduced light at 4 8C for 10 min. Samples were analyzed at 540 nmin a spectrophotometer. The protein level of each sample wasdetermined using the Lowry method (Lowry et al., 1951). The datawere calculated as mM of nitrite per mg of protein.

2.5. Histological examination

After behavioral experiments, the rats (n = 6) were anesthetizedand perfused with 200 mL of 4% paraformaldehyde. The brainswere removed and postfixed in the same fixative solution for 48 hand then were embedded in paraffin. Coronal 5-mm thick sectionswere taken from each brain at the level of the anterior temporalarea approximately �3.8 to �6.8 mm from bregma.

2.6. Amino-cupric-silver stain

Neurodegeneration caused by the injection of Ab into the TCxwas observed according to the precipitation of the ionic-silverstaining method (De Olmos et al., 1994). The preimpregnationsolution contained cupric nitrate, silver nitrate, cadmium nitrate,lanthanum nitrate, pyridine (Sigma–Aldrich, St. Louis, MO, USA.),and deionized water. After the components were well mixed, thesolution was warmed until it reached 45–50 8C. Sections wereincubated in this solution overnight. The impregnation solutioncontained silver nitrate, ethanol, acetone, lithium hydroxide,ammonium hydroxide (Sigma–Aldrich, St. Louis, MO, USA), anddeionized water. Sections were rinsed in deionized water, then inacetone, and then placed into the impregnation solution andincubated in this solution for 50 min. The reducing solutioncontained ethanol, formalin, citric acid (Sigma–Aldrich, St. Louis,MO, USA), and deionized water. The sections were transferred fromthe impregnation solution into the reducing solution and placed ina water bath with a constant temperature between 32 and 35 8C.After 25 min in the reducing solution, the sections were transferred

I.D. Limon et al. / Neuroscience Research 63 (2009) 129–137 131

into deionized water and rinsed twice. The sections were thencounterstained with neutral red (Sigma–Aldrich, St. Louis, MO,USA). Then sections were transferred into alcohol and xylene tofinally mount them permanently with Entellan (Merck Darmstadt,Germany). The neurons stained in the TCx and the CA1 of the Hpwere observed through a Leica DM/LS microscope at 40� (LeicaMicrosystems, Wetzlar, GmBH) and photographed using a digitalcamera Leica DFC-300FX (Leica Microsystems Digital Imaging,Coldhams Lane, Cambridge, UK). The digitized images werecaptured at the same level of contrast and sharpness using theIM1000 software (Imagic Bildverarbeitung AG, Leica Microsys-tems, Heerbrugg, Switzerland) and transformed into JPG files forstorage and analysis.

2.7. Hematoxylin and eosin (H&E)

The neurons were observed through a Leica DM/LS microscopeat 40�. Undamaged neurons were recognized as cells with roundblue nuclei and clear perinuclear cytoplasm. These were evaluatedusing the criteria formulated by Garcia et al. (1995). Damagedneurons were cells with changed nuclei (pyknosis, karyorrhexis,and karyolysis) and cytoplasmatic eosinophilia or loss of affinityfor hematoxylin. The number of undamaged neurons wasexamined in the TCx and CA1 of the Hp (Kiyota et al., 1991).The neurons were counted using images captured with digitalcamera Leica DFC-300FX (Leica Microsystems Digital Imaging,Coldhams Lane, Cambridge, UK) and using Leica software IM1000(Imagic Bildverarbeitung AG, Leica Microsystems, Heerbrugg,Switzerland). From all pictures we analyzed the undamagedneurons per optical field from four serial sections for each rat.

2.8. Immunohistochemistry

Paraffin was removed from sections (5-mm thick) and theywere rehydrated according to conventional histological techni-ques. Then they were rinsed with a phosphate-saline buffer (PBS,50 mM sodium phosphate, 0.15 M NaCl, pH 7.4). Nonspecificbinding sites were blocked by incubating in IgG-free 2% bovineserum albumin (BSA, Sigma) for 30 min. Afterwards, specimenswere incubated for 10 min with 0.2% Triton X-100 in PBS at roomtemperature. Then slides were rinsed with PBS. Slides were thenincubated overnight at 4 8C with a monoclonal mouse antibodyanti-nNOS and a polyclonal rabbit antibody anti-iNOS (Santa CruzBiotechnology Inc., CA, USA), a polyclonal rabbit antibody anti-glial-fibrillary-acidic protein (GFAP, Dako, Denmark A/S), andmonoclonal mouse antibody anti-3-nitrotyrosine (Santa CruzBiotechnology Inc., CA), all at 1:100 dilutions. The neuronsimmunostained in the TCx and the CA1 of the Hp were observedthrough a Leica DM/LS fluorescent microscope at 40� (LeicaMicrosystems, Wetzlar, GmBH). Fluorochromes were visualizedwith their specific filters and analyzed in three channels. Antibodylabeling was recognized with an isospecific secondary FITC-conjugated antibody and visualized in the green channel. Alsoantibody labeling was recognized with isospecific secondaryrodamine and visualized in the red channel. Slides were counter-stained with VectaShield with DAPI (Vector Labs., CA, USA) fornuclei staining (blue channel). The slides used were from near thesite of the injection for qualitative assessment. The images wereprojected with a Leica IM1000 version 1.20 release-9 computer-based program (Imagic Bildverarbeitung AG, Leica Microsystems,Heerbrugg, Switzerland).

2.9. Statistical analysis

Data were expressed as the mean � S.E. of each group used.Statistical analysis of data was made by two-way ANOVA for

the training and memory test. The number of crossings on theplatform quadrant, and the number of crossings on the oppositequadrants were analyzed using a Student’s t-test. The numbers ofneurons by H&E staining and nitrite levels were analyzed in thesame way. A level of P < 0.05 was accepted as statisticallysignificant.

3. Results

3.1. Effects on spatial learning and memory of Ab(25–35) injected into

the temporal cortex

Animal-training performance was examined 20 days afterinjection of Ab(25–35) by using the Morris water maze. The timeto find the platform of both the control and the Ab(25–35)-treatedgroup decreased over the course of the acquisition training. Thetime to find platform of the Ab(25–35) group increasedsignificantly, from the first to the 5th day of training, comparedto the control group. After 10 days of training the memorytest was examined using four trials. Fig. 1A shows that thetime to find the platform were significantly greater in theAb(25–35)-treated group than in the control group (memory testat day 30, ANOVA P < 0.001). Consequently, animals treatedwith Ab(25–35) spent more time because they made morecrossings to the opposite quadrants of the platform, whichactions were significantly different from the control group(P < 0.05) (Fig. 1B). Furthermore, the Ab(25–35)-treated groupmade less crossings to the target quadrant than did the controlgroup (P < 0.05) (Fig. 1C). The rats treated with Ab(25–35) spentmore time finding and reaching the hidden platform (81%) thanthe control rats.

3.2. The Ab(25–35) injected into the temporal cortex increases NO

production and causes cell loss in the temporal cortex and

hippocampus

The release of NO was evaluated by measurement of its stablemetabolite (NO2

�) in the homogenized temporal cortex andhippocampus 30 days after the 100 mM injection of Ab(25–35). InFig. 2A is shown the average of nitrites in the TCx of theAb(25–35)-treated group (7.89 � 1.03 mM/mg of protein) com-pared to the control group (5.04 � 0.45 mM/mg of protein). Wefound an increase of nitrites in the TCx of the Ab(25–35)-treatedgroup (57%) compared to the control group; Student’s t-testP = 0.026. Shown in Fig. 2C is the average of nitrites in the Hp of theAb(25–35)-treated animals (6.24 � 1.32) compared to the controlgroup (2.76 � 0.39), which was significantly different (126%);Student’s t-test P = 0.0002.

The effects to the Ab(25–35)-treated group on the number ofneurons was assessed using hematoxylin and eosin stains. Theslices showed eosinophilic neurons were more evident in the TCxand the CA1 of the Hp from the Ab(25–35)-treated group comparedto the control group. Some cells with pyknotic nuclei were alsopresent (not illustrated). In Fig. 2B and D we show the quantitativedata on neuronal densities in the TCx and the CA1 subfield of theHp, respectively. In the temporal cortex the density of neurons inthe control group (14 � 1) was different than the Ab(25–35)-treatedgroup (11 � 1), which represents a 20% loss of neurons caused byAb(25–35) injection; Student’s t-test P = 0.0264. The average ofneurons in the CA1 of the Hp from the control group was significantlygreater (70 � 3) than in the Ab(25–35)-treated group (27 � 7), whichrepresents a 61% loss of neurons caused by Ab(25–35); Student’s t-testP = 0.0002. This indicates that in the Hp there is a major loss ofneurons in Ab(25–35)-treated animals even when the injection wasmade into the TCx.

Fig. 1. Effects of Ab(25–35) injection into the temporal cortex on water-maze

memory in rats. In (A) shows the time to find the platform in the water maze during

the training and memory test. In (B) shows the number of crossings on the opposite

quadrant(s) during the memory test. In (C) shows the number of crossings on the

target quadrant. The memory test was made 30 days after the injection of 1 mL of

Ab(25–35) [100 mM] or 1 mL of vehicle (n = 13 for each group). The values represent

the mean of the time to find the platform or the number of crossings on the

quadrants � S.E. They were analyzed using a two-way ANOVA P < 0.001 in (A) and a

Student’s t-test P < 0.05 in (B) and (C) compared to the control group.

I.D. Limon et al. / Neuroscience Research 63 (2009) 129–137132

3.3. The Ab(25–35) injected into the temporal cortex increased

neuronal damage, 3-nitrotyration, and GFAP immunoreactivity in the

temporal cortex and hippocampus

The effects on the Ab(25–35)-treated group of neuronal damageof the TCx and the CA1 of the Hp neurons were studied with amino-cupric-silver stain. We found an evident argyrophilic reaction thatcharacterizes the neuronal damage in the TCx and the CA1 subfieldof the Hp in the Ab(25–35)-treated group (Fig. 3B and D). This wasdisplayed by the notably dark neuronal perikarya in dendrites and

axons from the Ab(25–35)-treated group compared to the lessernumber of dark cells in the same areas of the control group, inwhich damaged neurons were rarely found in the TCx and Hp(Fig. 3A and C).

The immunoreactivity to 3-nitrotyrosine (3-NT) and the glial-fibrilar-acidic protein (GFAP) were investigated as markers for theformation of reactive nitrogen species (RNS) and astrocyte activity.We found that GFAP immunoreactivity (red color) was found inlarge amounts and widely distributed in the region of TCx near thesite of the Ab(25–35) injection. In addition, the 3-NT fluorescence(green color) was found in almost all the neuronal cells, so it wasclear that 3-NT had a high immunofluorescence in the TCx and thiswas different from the controls that did not show an immunor-eactivity (Fig. 3E and F). Furthermore, in the CA1 subfield of the Hp,GFAP immunoreactivity was found uniformly, whereas the 3-NTwas located only in some few regions of CA1 as a disseminated andheterogeneous fluorescence in pericellular areas in the Ab(25–35)-treated group. Almost no immunoreactivity to 3-NT and GFAP wasfound in the control group for the same areas of the Hp (Fig. 3G andH). The nucleus was observed with DAPI staining (blue color).Taken together, these observations strongly support that theinjection of Ab(25–35) causes neuronal damage by NO in the TCx andthe CA1 of the Hp.

3.4. The Ab(25–35) injected into the temporal cortex increases NOS1

and NOS2 in the temporal cortex and hippocampus

To determine the effects of the Ab(25–35) injection on nNOS andiNOS immunoreactivity in the TCx and the CA1 of the Hp, tissueswere examined. We observed immunoreactivity to nNOS (greencolor) was more intense in the neuronal cells and uniformlydistributed in the TCx of Ab(25–35)-treated group than in the controlgroup, which showed less intense and little immunoreactivity tonNOS in the neuronal cells (Fig. 4A and B). The nNOS immunor-eactivity found in the CA1 subfield of the Hp for the Ab(25–35) groupwas more intense than in the control group in the same area(Fig. 4C and D). The results show that nNOS was negative in the TCxand the CA1 of the Hp of the control group (Fig. 4A and C), whereasthe inmunoreactivity to nNOS was more intense in the Ab(25–35)-treated group for the same areas (Fig. 4B and D).

In contrast, a high-power immunofluorescence to iNOS (greencolor) was observed. The cells show a marked and homogeneusimmunoreactivity in the TCx and the CA1 subfield of the Hp causedby the injection of Ab(25–35) (Fig. 4F and H). Almost all the bodycells stained intensely to iNOS. Our results showed very clearlythat iNOS was present in the TCx and Hp, whereas the controlgroup had less immunoreactivity to iNOS in the same areas of thebrain (Fig. 4E and G). The nucleus was observed with DAPI staining(blue color). The results of our study showed that increased iNOSand nNOS immunostaining were seen in neurons from the TCx andthe CA1 of the Hp for the Ab(25–35)-treated group.

4. Discussion

AD is predominantly a disorder that results in total dementiaassociated with the neurodegenerative process in several brainregions, such as the hippocampus and cortex. Because the cerebralcortex shows the pathological changes characteristic for AD, itappears that the neurodegeneration spreads out from there (Braakand Braak, 1991, 1998).

The Ab(25–35) fraction could be a useful tool in an animal modelto reproduce the neurotoxic effects similar to those produced byAb(1–42) and the impairment of the memory (Maurice et al., 1996;Delobbette et al., 1997; Yamada and Nabeshima, 2000; Yamaguchiand Kawashima, 2001; Stepanichev et al., 2004; Perez-Severianoet al., 2004; Cheng et al., 2006). In agreement with these findings,

Fig. 2. Effects of Ab(25–35) injected into the temporal cortex on the measured nitric oxide and the neuronal loss in the TCx and Hp of rats. In (A) and (C) the graph shows nitrite

levels for the TCx and Hp of the control and the Ab(25–35) group. The NO was measured as nitrites by the Griess method and the protein by the Lowry method after the

behavioral test was finished. The values represent the mean of nitrites [mM]/mg of protein � S.E. *P < 0.05 with the Student’s t-test. In (B) and (D) the brain sections were stained

with H&E to show the number of neurons in the TCx and Hp of the Ab(25–35) group or control group observed at 40�. The values represent the mean number of neurons � S.E.

(counts/500 mm). Student’s t-test P < 0.05 vs. the control group (n = 7 in each group).

I.D. Limon et al. / Neuroscience Research 63 (2009) 129–137 133

we assessed whether the injection of the Ab(25–35) fraction into thetemporal cortex causes the neurodegeneration process through NOpathways and impairs the spatial memory of rats. Our findings ofthe effects of Ab(25–35) on the cognitive process of animals show aloss of memory in the water maze 1 month after the injection,which is related to the high activity of NO pathways. Our resultssuggest that NO exerts an important source of neuronal damage.

The question to be resolved is the role of Ab(25–35) in thetemporal cortex and its implications on spatial memory. Our studyshows that spatial learning and memory were delayed in animalsinjected with Ab(25–35) compared to the control group because thetimes to find the platform were increased. Furthermore, Ab(25–35)-treated animals had a larger number of crossings to the oppositequadrants during the memory test and so spent more time findingthe platform. We did observe that both groups had a similarperformance during the beginning of the test, with the trajectorythat they followed in the first trials. During the progress of thetrials the animals with Ab spent more time to find the platform,but they can swim with the same skill as the control group. Inaddition, we found no difference in swimming speeds between thegroups. These findings indicate that injection of Ab(25–35) into thetemporal cortex causes impairment of spatial memory in rats after1 month, as would be expected. These data indicate that injectionof Ab(25–35) causes a cognitive impairment and neuronal damage.Although other experiments from different research groups haveshowed the inhibition of long-term potentiation (LTP) andimpairment of learning evaluated in the water maze 3 days afterthe injection (Sun and Alkon, 2001).

Microscopic observations after H&E and aminocupric stainingslead to the assessment that there were both cytoarchitectural anddegenerative changes in neurons in the regions of interest, such asthe CA1 of the hippocampus and the temporal cortex. We founddamaged neurons that appear as a loss of hematoxylin affinity inthe Ab(25–35)-treated group (not illustrated). We also found asignificant decrease of neurons in the temporal cortex and the CA1of the hippocampus. The aminocupric staining there appears mostintense, which indicates an increase of an argyrophilic reactionthat correlates with neuronal damage. These effects suggest thatAb(25–35) is able to cause neuronal alterations in both the temporalcortex and hippocampus that could be correlated with a functionalimpairment, such as memory deficit.

The possible involvement of reactive oxygen species (ROS) inthe deposition of fibrillar Ab and its neurotoxicity is a concept thathas been discussed for several years (Behl, 1999; Velliquete et al.,2005). Several studies have associated oxidative stress withneuronal injury and cognitive impairment (Cutler et al., 2004).Moreover, oxidative modifications have been found in cerebraltissue in the early stages of AD, whereas oxidation of nucleic acids,proteins, and lipids are all found in the early changes in human ADas well as in cellular and animal AD models (Smith et al., 2000).

Our study also showed there are higher nitrite levels in theAb(25–35)-treated group compared to the control group when weexamined the temporal cortex and hippocampus. These findingssuggest that Ab(25–35) is responsible for the activation of themachinery of NO production. Note that NO is an important sourceof ROS, such as O2

� and ONOO�, which appear to share an

Fig. 3. Effects of Ab(25–35) injected into the temporal cortex enhances neuronal damage, 3-NT, and GFAP of the TCx and Hp of rats. Photomicrography of aminocupric stain

shows the neuronal damage and the morphologic alterations in the TCx and the CA1 subfield of the Hp. The rats were injected with 1 mL of Ab(25–35) [100 mM] (n = 6, in (B),

(D), (F), and (H)) or 1 mL of vehicle (n = 6, in (A), (C), (E) and (G)). The locations of nitrated protein (green color) and GFAP (red color) were examined in the TCx and the CA1

subfield of the Hp of animals treated with Ab(25–35) (F and H) and control animals (E and G). The nucleus was observed with DAPI staining (blue stain). These evaluations were

done 30 days after the injection of Ab(25–35) and all stains were observed 40�.

I.D. Limon et al. / Neuroscience Research 63 (2009) 129–137134

important role in the neurodegenerative process and neuronaldeath. There appears to be a strong correlation between region-specific NO formation and specific, persistent damage to cyto-chrome-c oxidase in AD (Parks et al., 2001).

The increase of NO levels promotes protein nitration in differentareas of the rat brain injected with Ab(25–35), similar to that whichhappens in the brain of patients with AD (Sultana et al., 2006).These results suggest that NO overproduction causes the produc-tion of other highly toxic reagents like ONOO�, which enhance the

neuronal damage by a profound disruption of energy metabolism(Mattson, 2004). This may be relevant for the understanding of thepathogenesis of this disorder. Large amounts of NO can beproduced by activated astrocytes and microglia after induction ofiNOS. Incubation in vitro of cortical astrocytes with Ab causes iNOSinduction (Hu et al., 1998) through two pathways, the transcrip-tion factor kappa B (NFkB)-dependent mechanism (Akama et al.,1998) or by microglial activity (Parks et al., 2001; Moncada andBolanos, 2006).

Fig. 4. The injection of the Ab(25–35) fraction into the temporal cortex increases NOS immunoreactivity in the TCx and Hp of rats. The rats were injected with 1 mL of Ab(25–35)

[100 mM] or 1 mL of vehicle (n = 6). Thirty days after the injection the nNOS or iNOS were measured by using immunofluoresce staining. Photomicrography of

immunoreactivity to nNOS of the TCx (A and B) and hippocampus (C and D) observed at 40�. The immunoreactivity to iNOS from the TCx (E and F) and the CA1 subfield of the

Hp (G and H) were analyzed. Anti-nNOS (red color), anti-iNOS (green color), anti-GFAP (red color), and DAPI (blue stain).

I.D. Limon et al. / Neuroscience Research 63 (2009) 129–137 135

There is also evidence in the brain tissue of patients with ADthat iNOS is expressed (Heneka et al., 2001; Haas et al., 2002). Ourresults show that the Ab(25–35) fraction increased iNOS greaterthan nNOS expression, simultaneously with the GFAP activated inreactive astrocytes in the temporal cortex and hippocampus. Thisappears to be related to overproduction of NO found in the Ab(25–

35)-treated animals similar to that which happens in the brains ofpatients with AD, in which there is an accumulation of Ab plaquesthat activate astrocytes and microglia (Akama et al., 1998). Thecommon pathological hallmarks of several neurodegenerativediseases include the loss of neurons associated with or followed by

massive activation of astrocytes (Sriram et al., 2004). Recently,because a model of AD-type neurodegeneration showed that eNOSwas overexpressed and accompanied by oxidative stress, mito-chondrial dysfunction, activation of several proapoptotic mechan-isms, neuronal loss, and gliosis in the cortex of rats (De la Monteet al., 2007), then it is probable that eNOS may also cause neuronaldeath by Ab(25–35) injection.

Despite evidence that NO and ONOO� are important factors inthe oxidative stress, in the brain tissue from patients of Alzheimer’sdisease it has been found that nitrotyration is increased in theneurons and glia of the cerebral cortex, which suggests that

I.D. Limon et al. / Neuroscience Research 63 (2009) 129–137136

nitrotyration plays a key role in the neurodegenerative process ofAD (Smith et al., 1997). The inflammatory response has beenreported as an effect of NO production, which is seen in AD tissue asincreased amounts of nitrotyrosine-modified proteins (Good et al.,1996; Smith et al., 1997). The response to the Ab fraction is toincrease NOS expression. The first to occur is nNOS with theintention of repair, then followed by iNOS, which causes higher NOlevels and serious damage to cells by the production of otherreactive nitrogen species and even nitration of proteins (Rodrigoet al., 2004). In our experiments, the Ab(25–35)-treated animalsshowed a greater immunoreactivity to 3-NT proteins whencompared to animals without Ab(25–35). We suggest that Ab(25–

35) may be a useful model of AD as a cause of neurodegeneration inneurons through the production of NO and the upregulation ofGFAP, nNOS, iNOS, and the nitration of proteins. NOS and 3-NTwere not enhanced in the presence of L-NAME (an inhibitor of NOS)before Ab(25–35) injection (data not shown).

It appears that under oxidative stress there is a dysregulation ofiron homeostasis with an insufficient antioxidant response,because Ab deposits could be caused by increased iron andhemeoxygenase-1 (HO-1) by abnormalities in the mitochondrialturnover that promotes oxidative stress via the increase of redox-active iron in the pathology of AD (Sayre et al., 2008). These effectscause metal accumulations, which are major producers of ROS,accompanied by oxidation biomacromolecule types, such as DNA,RNA, and proteins, and lipoperoxidation (i.e. malondialdehyde(MDA) or 4-hydroxy-2-nonenal) (Sayre et al., 2005). It is alsopossible that these markers are involved in the toxicity in rats withinjection of Ab(25–35).

Moreover, it appears that oxidative damage precedes Abdeposition in AD as the models of AD have shown (Nunomuraet al., 2001, 2000). Studies in cell culture and transgenic animalsof AD have shown that increased lipid peroxidation, proteinoxidation, and decreased copper–zinc superoxide dismutase(SOD) activity precede Ab deposition or Ab-fibril formation inthe transgenic mouse and Caenorhabditis elegans models of ADamyloidosis (Pratico et al., 2001; Drake et al., 2003; Schuesselet al., 2005). High concentrations of Ab in the micromolar rangecan lead to oxidative stress in several biologic systems, such aswe have found with the injection of Ab(25–35) into the rat. Incontrast in vitro and in vivo studies have demonstrated aneuroprotective effect from the antioxidant activity of Abbecause it is associated with a decrease in oxidative stress (Leeet al., 2006). Physiological concentrations (in a low nanomolarrange) of Ab(1–42) have been shown to protect the culturedneurons from iron- and copper-caused toxicity and to protectlipoproteins from oxidation in the cerebrospinal fluid and plasma(Zou et al., 2002; Kontush et al., 2001). In addition, other studieshave shown that generation of hydroxyl radicals by free Cu(II) isdecreased in the presence of Ab(1–42) by its three histidineresidues that control the redox activity of this metal. Ab exertsan antioxidant activity under these circumstances that protectsagainst lipoperoxidation (Hayashi et al., 2007; Nakamura et al.,2007). Therefore, it appears Ab has a neuronal protectiveresponse against oxidative stress, though it is not clear whenit becomes toxic. This may depend on the aggregation state of theprotein and the amount of Ab and other factors (Nunomura et al.,2006, 2007).

Our study clearly shows that the Ab(25–35) fraction injected intothe temporal cortex impairs the long-term spatial memory ofanimals, caused by the overproduction of NO. At the same time wemeasured an increase of the expression of nNOS and iNOSaccompanied by astrocytosis along with important alterations ofneurons such as nitration of proteins in both the temporal cortexand hippocampus of rats. This finding represents novel resultsabout the role of Ab(25–35) in the temporal cortex of rats.

Acknowledgements

This work was supported in part by CONACYT (61205), INNN(15/06), UNAM PAPIIT IN214609 and IN212108 given to J.G., andthe Laboratory of Neuropharmacology-BUAP grant (LNAB1-08)given to D.L. Thanks to Dr. Ellis Glazier for editing this English-language text.

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