INTRODUCTION
ementia is an organic brain disease defined as a
loss of intellectual ability of sufficient severity to interfere with either
the occupational functioning or usual social activities. Prevalence of dementia
increases exponentially with advancing age, ranging from 10% in the age group
of 60-65 years to 38.6 in the age group of 90-95 years [1]. The dementing
condition that has received the utmost attention is Alzheimer’s disease. Unlike
other dementias, Alzheimer’s is a progressive neurodegenerative condition [1]. A
number of environmental factors have been put forward as possible contributory
cause of Alzheimer’s disease in some people and aluminum is identified as one
of these factors [2].
Aluminum has
been implicated in human neurodegenerative diseases like Alzheimer’s. Possible
mechanisms of aluminum induced neurotoxicity have been related to cell damage
via free radical production. Increased lipid peroxidation (LPO) is the major
consequence associated with oxidative stress. Aluminum has been shown to be
associated with both plaques and tangles in the Alzheimer. Various
investigations have suggested that Alzheimer’s disease is more common in areas
where the aluminum content in water supplies is the highest [3, 4].
Several
antioxidants have been studied for the reduction of oxidative stress occurring
during Alzheimer’s disease [5, 6]. Non-steroidal anti-inflammatory drugs
(NSAID) are the group of drugs which work by interfering with the
cyclooxygenase pathway. NSAID have been associated with antioxidant property
and have been shown to improve the circulating antioxidant status on daily
dosing in rheumatoid arthritis [7, 8].
Meloxicam
is a NSAID that appears to have a greater selectively inhibitory activity
against the inducible cyclooxygenase-2 (COX-2) isoform, than against the
constitutive isoform cyclooxygenase-1 (COX-1). Inhibition of COX-1 is
associated with gastrointestinal, renal, and platelet aggregation adverse
effects. Therefore, meloxicam and other COX-2 selective inhibitors are promoted
for their safer profile of side effects. Meloxicam also has been shown to
possess significant antioxidant property [9]. The objective of the present
study was to investigate the anti oxidant role of meloxicam in aluminum induced
oxidative stress in rat brain.
MATERIALS
AND METHODS
Reagents. Aluminum
chloride (AlCl3) was obtained from S.d Fine Chemicals Ltd,
Mumbai, India and Meloxicam injections
(5 mg/mL) were a gift from INTAS Pharmaceuticals, Ahmedabad, India.
Animals. Sprague Dawley rats (n =
30), weighing 184.64 ± 5.81 g, of either sex bred in the animal colony at the
B.V. Patel PERD, Centre, Ahmedabad, from the original stock obtained from
National Institute of Nutrition, Hyderabad, were used for the study.
Housing
conditions. The animals were housed 3 per cage in
polypropylene cages and were moved to the experimental room where they were
allowed to acclimatize for a day before treatment. The environmental conditions of the animal
room were as per a specific design. A 10% air exhaust in the air conditioning
unit was maintained along with a relative humidity of 60 ± 5% and a temperature of
25 ±
3oC were stabilized. A 12 hour light/dark cycle was also regulated
for the experimental animals. Amrut certified rodent diet (Maharashtra Chakan
Oil Mills Ltd., India) and tap water (boiled
water cooled to room temperature) were provided ad libitum to the experimental animals.
All
experimental protocols were reviewed and accepted by the Institutional Animal
House Ethics Committee prior to the initiation of the experiment. Animals were
divided into the following treatment groups: 1. Control (double distilled water)
2. Aluminum Treatment 3. Meloxicam Treatment 4. Aluminum and Meloxicam treatment.
All the experiments were conducted both in vivo and in vitro.
In vivo treatment. Animals in all the groups were dosed for 28 days
as per the above treatment (n = 6 per group). Double distilled water and aluminum
(4.2 mg/kg body weight aluminium chloride) were dosed by i.p. injections [10]
whereas meloxicam (5 mg/ kg body weight) was dosed by intra muscular route.
Animals were sacrificed on the 29th day by euthanasia. Brain was
removed and washed with saline to remove blood and divided into two halves. One
half was transferred to HBSS (pH 7.4) and the second half chilled Tris buffer
50mM (pH 8.2), for the estimation of LPO [11] and assay of superoxide dismutase
(SOD) activity [12], respectively.
In vitro treatment. Previously, untreated animals (n = 6) were
sacrificed and the brains were excised, divided into two equal halves and
transferred into the respective buffers for LPO and SOD estimations as mentioned
above. The tissues were minced thoroughly and each half was further divided
into four equal parts. First part was treated with double distilled water,
second part was treated with aluminum chloride (4 µg/ml), the third part was
treated with meloxicam (5 µg/ml) and the fourth part was treated with aluminum
and meloxicam (4 µg/ml and 5 µg/ml, respectively). The treated minced tissues
were incubated for 20 minutes and were then estimated for LPO and SOD activity.
Estimation of LPO. The brain tissue was taken in 2 ml HBSS and was
homogenized at 3000 rpm using Polytron homogenizer (three cycles 30 seconds
each). The homogenate was then centrifuged at 710 ´g for 10 minutes. The
pellet was resuspended in 1 ml of HBSS that was then used for the estimation of
LPO. LPO was measured in terms of malonyl-aldehyde (MDA): thiobarbituric acid
(TBA) reaction by Okhawa et al. [11]. The reaction mixture contained 0.1
ml of tissue homogenate, 0.2 ml of sodium dodecyl sulfate, 1.5 ml of acetic
acid and 1.5 ml of aqueous solution of TBA. The pH of 20% acetic acid was
pre-adjusted with 1 M NaOH to 3.5. The mixture was made up to 4 ml with
distilled water and heated at 95ºC for 1 hour, in a water bath. After
cooling, 1 ml of distilled water and 5 ml of mixture of n-butanol and pyridine (15:1)
were added and mixture was shaken vigorously on a vortex mixer. After
centrifugation at 450 ´g for 10 min the amount of MDA formed was
measured by absorbance of the upper organic layer at 532 nm.
Animals loaded with
aluminum developed brain lesions, that is similar to those found in Alzheimer’s
disease [13, 14]. Aluminum uptake in the
brain does not depend on the alterations to blood brain barrier (BBB)
permeability. Transport of aluminum from brain interstitial fluid (BIF) to
blood is believed to be mediated by a monocarboxylate transporter located in
BBB. Several processes like endocytosis, facilitative diffusion, active
transport are implicated in the uptake of aluminum and its relative contri-butions
in each cell type in each brain region [13].
The
proteolytic cleavage of the amyloid precursor protein and the production of
amyloidogenic beta amyloid peptide are believed to be critical events in the etiology
of Alzheimer’s disease. Aluminum is bound by the beta amyloid and is found co-localized
with beta amyloid in the Alzheimer’s disease brain [15].
Amyloid
fibrils formed in the presence of aluminum are slightly thicker, significantly
longer and spirally wound around each other. Subsequent studies have been
conducted to confirm that Amyloid beta sheets (Aβ) (1-40) will bind up to
4 aluminum atoms and that binding increased the β-sheet content of the
peptide [16]. The neurotoxicity of Aβ in whatever form may involve the
formation of reactive oxygen species. Aluminum is a pro-oxidant and is known to
promote the oxidation activity of Aβ in the presence of iron. Aluminum has
also been linked to Aβ production through the immune response. It is also
linked to activate complement which in turn has been linked to the enhanced
aggregation of Aβ. [15]
One
group of brain cells in which aluminum is found are the glial cells. Their
primary function in the brain is maintenance of the short term constancy of
BIF. Glial cells respond to brain toxins by increased expression of glial
fibrillary acidic protein. Disruption of glial cell function by aluminum could
lead to accumulation of unwanted, possibly cytotoxic debris as well as
modulation of synaptic transmission and neuron-glial signaling. Exposed glia
does accumulate aluminum and show cellular changes indicative of oxidative
stress [15]. Active oxygen is known to cause many lesions. Mechanism of the
inhibition of active oxygen species appear to be important for the treatment of
disease. Recently, many investigators have studied the ability of various drugs
to eliminate active oxygen in particular.
The
mechanism of aluminum neurotoxicity has been postulated as a possible
contributing factor in several neurodegenerative diseases such as Alzheimer’s
disease [17]. In the current study, the group of animals treated with aluminum
chloride expressed high MDA levels which suggest that aluminum induces
superoxide anion radicals in the rat brain cells both in vivo and in
vitro. NSAID have been shown to have anti-oxidant like properties [8].
Although, the exact mechanism of action is yet not known, many investigators
have shown that these NSAIDs eliminate the active oxygen species, superoxide
anion radical in particular [8]. In the
current study, the role of meloxicam in reducing free radical load in the aluminum-treated
animals was studied. Significant decrease in the MDA levels was seen in
meloxicam- and aluminum-treated groups both in vivo and in vitro.
Meloxicam, a preferential COX-2 inhibitor
showed reduction in MDA levels in the brain. Meloxicam has been reported to be
able to cross the BBB [18] and has also been reported to reduce oxygen radical
generation in rat gastric mucosa [19]. No inhibition was observed in SOD levels
in brain and in the circulating levels with aluminum or meloxicam treatment.
However, we had earlier shown that circulating SOD levels were improved with
NSAID treatment in rheumatoid arthritis [7].
Decrease in the MDA levels with meloxicam in aluminum-treated animals is an important
finding which can be studied further for Alzheimer management.
ACKNOWLEDGEMENTS
The
authors are thankful to Gujarat Council for Science and Technology and
Industries Commis-sionerate, Government of Gujarat (India) for financial support.
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