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Meneghini et al 2009

Meneghini et al 2009

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Published by: Érica Engrácia Valenti on Dec 27, 2010
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921
CLINICS 2009;64(9):921-6
BASIC RESEARCH
I
Departamento de Clínica Médica, Disciplina de Cardiologia, Faculdade deMedicina do ABC - Santo André/SP, Brasil.
II
Departamento de Medicina, Disciplina de Cardiologia, Universidade Fe-deral de São Paulo (UNIFESP) - São Paulo/SP, Brazil.
III
Departamento de Saúde Pública, Universidade de São Paulo - São Paulo/ SP, Brasil.Email: celsoferreira.dmed@epm.brTel: 55 11 4993.5403Received for publication on May 18, 2009Accepted for publication on June 24, 2009
MEMANTINE PREVENTS CARDIOMYOCYTESNUCLEAR SIZE REDUCTION IN THE LEFTVENTRICLE OF RATS EXPOSED TO COLD STRESS
Adriano Meneghini,
I
Celso Ferreira,
I, II
Luiz Carlos de Abreu,
III
Vitor E.Valenti,
II
Marcelo Ferreira,
I
Celso F. Filho,
I
Neif Murad
I
 doi: 10.1590/S1807-59322009000900014
Meneghini A, Celso Ferreira, Luiz Carlos de Abreu, Vitor E. Valenti, Marcelo Ferreira, Celso F. Filho, Nei Murad. Memantineprevents nuclear size reduction in cardiomyocytes in the let ventricle o rats exposed to cold stress. Clinics. 2009;64(9):921-6.
OBJECTIVES:
Memantine is an N-methyl-d-aspartate (NMDA) glutamate
 
receptor antagonist used to treat Alzheimer’s disease.Previous studies have suggested that receptor blockers act as neuroprotective agents; however, no study has specically investigatedthe impact that these drugs have on the heart. We sought to evaluate the eects o memantine on nuclear size reduction in cardiaccells exposed to cold stress.
METHOD:
We used male EPM-Wistar rats (
n
=40) divided into 4 groups: 1) Matched control (CON); 2) Memantine-treated rats(MEM); 3) Rats undergoing induced hypothermia (IH) and 4) Rats undergoing induced hypothermia that were also treated withmemantine (IHM). Animals in the MEM and IHM groups were treated by oral gavage administration o 20 mg/kg/day memantineover an eight-day period. Animals in the IH and IHM groups were submitted to 4 hours o hypothermia in a controlled environmentwith a temperature o - 8ºC on the last day o the study.
RESULTS:
The MEM group had the largest cardiomyocyte nuclear size (151 ± 3.5 μm
3
vs. CON: 142 ± 2.3 μm
3
; p<0.05), while theIH group had the smallest mean value o nuclear size. The nuclear size o the IHM group was preserved (125 ± 2.9 μm
3
) comparedto the IH group (108 ± 1.7 μm
3
; p<0.05).
CONCLUSION:
Memantine prevented the nuclear size reduction o cardiomyocytes in rats exposed to cold stress.
KEYWORDS
: Memantine; Cardiac myocytes; Cardiotonic agents; Cell nucleus structures; Heart.
INTRODUCTION
Reduced body temperature induced by exposure to acold surrounding environment is considered a physiologicalstressor.
1-3
Neurogenic lesions related to myocardialhypertrophy and changes in myocardial tissue metabolism
4
 can be also caused by the reaction o cardiovasculartissue to cold stress, and these lesions are hypothesizedto be a marker or subsequent cardiovascular disease aswell as a predictor o hypertension.
5
Recently, this modelwas used to investigate several aspects o cardiac injury.
6
 Interestingly, a previous study indicated that cold stressacutely induces a reduction in the nuclear size (henceatrophy) o cardiomyocytes in rats,
7
which is in contrastto the nding that is observed in myocardial hypertrophy.This reduction in nuclear size has been hypothesized to be amarker o ischemia during periods o physiological stress.
7
It has been well established that glutamate excitotoxicitytriggers
 
neurodegeneration in patients with medical conditionsthat can lead to acute brain injuries such as stroke,
 
statusepilepticus or head trauma. Drugs that block N-methyl-d-aspartate (NMDA) glutamate
 
receptors have been shownto be neuroprotective in animal models o these medicalconditions.
8,9
Memantine is a non-competitive antagonisto NMDA receptors and is currently used to treat patients
 
922
CLINICS 2009;64(9):921-6Memantine role in heart tissueMeneghini A et al.
with moderate to severe Alzheimer’s disease to improvecognition.
10,12
Its mechanism o action is thought to be relatedto its eects on Ca
2+
homeostasis. An increase in cytosolicCalcium (Ca
2+
) is associated with unctional impairmento many organelle and is also strongly associated withapoptosis.
13
Previous studies have provided evidence thatmemantine is able to prevent ischemic injuries in the retinaand in neurons exposed to dierent aggressive agents (thatlead to increases in cytosolic Ca
2+
) due its eects on Ca
2+
.
14,15
Although various studies have suggested that NMDAreceptor antagonists have neuroprotective properties, noprevious study has specically investigated the infuencethis class o drugs may have on the heart. Previous studies,however, have reported an association between memantineand the prevention o ischemia under conditions o cellularstress.
14,16
Thereore, we sought to evaluate the eects o memantine on the nuclear size o cardiomyocytes in the letventricle o rats exposed to cold stress.
METHODSAnimals
Experiments were conducted on orty adult male Wistarrats that weighed 200-250 g. The ambient temperature o the environment in which the rats were housed was 22ºC,with the humidity at ~60%. The rats were kept on a 12-hourlight/12-hour dark cycle. Animals had ree access to oodand water. Animals were randomized into our groups: 1) thecontrol group (CON, n=10), in which rats were treated withgavage administration o 1 mL o water or eight consecutivedays; 2) the memantine group (MEM, n=10), in which ratswere treated with gavage administration o water (1 mL)containing 20 mg/kg o memantine or eight consecutive days;3) the induced hypothermia group (IH, n=10), in which ratswere treated with gavage administration o 1 mL o water oreight consecutive days and exposed to a –8ºC environmentor our hours on the last (8
th
) day o the study and; 4) thememantine plus induced hypothermia group (IHM, n=10), inwhich rats were treated with gavage administration o water(1 mL) containing 20 mg/kg memantine or eight consecutivedays and exposed to a –8ºC environment or our hours on thelast (8
th
) day o the study. All experiments were perormedin accordance with the ethical guidelines o the NationalInstitutes o Health Guide or the Care and Use o LaboratoryAnimals and were approved by the Ethical Committee orresearch at our University (number 003/08).
Induced Hypothermia Procedure
Rats were exposed to cold stress, which was inducedby placing them in wire mesh cages in an open rerigeratedcompartment at -8ºC or 4 hours. Rats were exposed to thisenvironment only once and their behavior was observedthroughout the stress experiment
7
. Rats’ body temperatureswere monitored and maintained near 37ºC. No rats diedduring the induced hypothermia procedure.
Histological Examination
To veriy i the exposure to cold stress was sucient tocause stress responses characterized by lipid and glycogendepletion in adrenal gland and liver, we evaluated lipiddepletion in adrenal gland cortical cells and glycogen depletionin hepatocytes. Ater administration o an adequate level o ether anesthesia, we examined the rats’ tail tone and responseto external stimuli beore and during surgical procedure throughthe evaluation o vibrissa movements. All animals were then submitted to laparotomy. Two pieceso the let lobe o the liver and right adrenal gland wereremoved or microscopy investigation. These specimenswere cut into small pieces (1 mm
3
), post-xed in 1% OsO
4
 solution or 2 hours, dehydrated and embedded in araldite.Silver or gray thin sections (60-90 nm) were produced usinga Porter-Blum MT-B ultramicrotome. The sections were thenmounted on copper silver grids with 200 patches and stainedwith uranyl acetate and lead citrate. We presented dataregarding lipid depletion in adrenal gland cortical cells andglycogen depletion in hepatocytes, respectively, as stainingintensity levels (+=small intensity; ++++=high intensity).
6
Nuclear Volume Measurement
All animals were also submitted to thoracotomy. Thethorax o each rat was opened and the let ventricle wasexposed and removed. Fragments o heart material wereixed in Bouin’s solution, mounted and parainated.Sections measuring 10μm were stained with hematoxylin-eosin. In order to estimate the nuclear size o each cell,karyometry was used according to the same principlesdescribed in a previous publication.
17
Morphometricevaluation was perormed using the Quantimet Color Option(Leica, Cambridge) image analysis system. Measurementso cardiomyocyte nuclear parameters were perormedexclusively on clearly visible longitudinal sections o musclebers in which cardiomyocyte nuclei had a clear outline. Tocalculate nuclear volume, we used the ollowing equationproposed by Salvatore:
17
V= (A
2
x B) /1.91V=volume; A=smaller axial measure; B=bigger axialmeasure; 1.91=constant.
 
923
CLINICS 2009;64(9):921-6Memantine role in heart tissueMeneghini A et al.
Using a low power eld diameter o 1800 μm, outlineso 150 nuclei were drawn or each sample by camera lucida.Arithmetical means o the diameters o nuclei were groupedinto requency classes. The samples were examined by threeindependent investigators using standardized criteria.
7
Statistical Analysis
The results are reported as means ± standard deviations.In order to examine cell volume data, the Kruskal-Wallis and Tukey post-hoc tests were applied to allowcomparisons between independent groups. The concordanceo the measurements perormed by the three individualinvestigators was evaluated and analyzed using Bartko’sintra-class correlation coecient according to the Fleissguidelines.
17
The signicance level was set at R>0.75 orp<0.05.
Bartko’s
 
test formulaR-
Bartko’s correlation index
PMS-
Patients Mean Square
RMS-
Researcher Mean Square
EMS-
Error Mean Square
N-
Number o events
K-
Number o investigators
RESULTS
We noted the ollowing reponses in rats ater cold stressexposure: hair bristling, paw edema, tremor, tail stretchingand tachypnea. There were no response dierences notedbetween the memantine-treated group and the untreatedgroup. Body weights were not statistically dierent amongthe groups (IH group: 306.2±20.3 grams; CON group:308.6±29.7 grams; MEM group: 265.8±29.4 grams; IHMgroup: 254.6±21.9 grams).To validate whether or not exposure to cold stress wassuicient to cause stress response, we examined lipiddepletion in adrenal gland cells (Table 1) and glycogendepletion in hepatocytes (Table 2) o rats rom each group.The IH group had highest rate o lipid depletion inadrenal gland cells (p<0.05) and the highest rate o glycogendepletion in hepatocytes (p<0.05), which supports ourhypothesis that exposure to -8ºC or our hours was eectiveat inducing physiological stress.The rats in the IH group had a smaller cardiomyocytenuclear size (Figure 1) compared to the other three groups(p<0.05). Our analysis revealed that the rats in the IHMgroup had a larger nuclear size than rats in the IH group,likely because the memantine treatment provided protectionon the ultrastructural level (p<0.05).We compared teh groups on the basis o nuclear size(Figure 1). The nuclear size o the IH group (Figure 2A)was signicantly decreased as compared to the control group(Figure 2B) (p<0.05). Furthermore, rats in the IHM group(Figure 2C) had a 76% reduction in their cardiomyocytenuclear size as compared to rats stressing the MEM group(Figure 2D) (p<0.05). Data variance analysis using Bartko’s
 
correlation index yielded results ranging rom 0.44-0.96 inall experimental groups, thus validating our methodology.Furthermore, when we perormed variation analysis
Table 1 -
Lipid depletion intensity in adrenal gland corticalcells stained with Sudan IV* (+=small intensity; ++++=highintensity)
AnimalCold StressControlMemantineMemantine +Cold Stress
1
++++++++++++
 2
+++++++++++
 3
++++++++++
 4
++++++++++
 5
++++++++++++++
+++++++++
++++++++
8
+++++++++
 9
+++++++++++
10
+++++++++
* + = Low intensity (more lipid depletion); ++++ = High intensity (lesslipid depletion)
Table 2 -
Glycogen depletion intensity in hepatocytes blushedwith PAS method* (+=small intensity; ++++=high intensity)
AnimalCold StressControlMemantineMemantine +Cold Stress
1
++++++++++++
 2
++++++++++++
 3
++++++++++++
 4
+++++++++++
 5
+++++++++++
+++++++++++
+++++++++
8
+++++++++
 9
++++++++++
10
++++++++++
*+ = Low intensity (more glycogen depletion); ++++ = High intensity(less glycogen depletion)

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