Neuroprotection against Excitotoxicity and Oxidative Stress by

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					           Neuroprotection against Excitotoxicity and Oxidative Stress
                      by Hypothermia and Alkalinization

                            D.P. Kuffler, Ph.D. and I. Berrios, B.S.
                                  Institute of Neurobiology
                                  University of Puerto Rico

Corresponding author:
Damien Kuffler, Ph.D.
Institute of Neurobiology
210 Blvd. del Valle
San Juan, PR 00901

Tel: 787-721-1235
FAX: 787-725-1289

Short running title: Novel technique providing neuroprotection

Key Words: adult rat, dorsal root ganglia, hypothermia, alkalinization, ischemia, potassium
cyanide, neuroprotection

Two major forms of neurotoxicity are excitotoxicity and oxidative stress (ischemia).
Excitotoxicity causes neuron death by inducing a massive release of excitatory amino acids
(EAAs) causing neuron to be exposed to an excessive accumulation of EAAs. These EAAs cause
extracellular acidification, excess NMDA receptor activation, excessive calcium influx,
intracellular accumulation of calcium , the disruption of calcium homeostasis and neuron death .
Oxidative stress is exemplified by neurotoxicity induced by potassium cyanide in which neuron
death results primarily from the inhibition of cellular metabolism and mitochondrial disfunction,
but also due to loss of calcium homeostasis and extracellular acidification. Therefore, b ecause
some neurotoxic actions of oxidative stress and excitotoxicity are similar, neuroprotection
against one can be protective against the other. In this mini-review, we examine data about the
neurotoxic action s of excitoxicity and oxidative stress, and means of providing neuroprotection
against the neurotoxic influences of glutamate and cyanide. Finally, we present preliminary data
showing that both hypothermia and alkalinization separately, but to a greater extent when
combined, provide neuroprotection to adult rat dorsal root ganglion neurons against glutamate
and cyanide, and that their neuroprotection is enhanced when combined with adenosine. We
propose that hypothermia-induced neurop rotection is in part provided by reducing or blocking
NMDA receptor activation and that alkalinization -induced neuroprotection is by blocking
neurotoxic extracellular acidification and by precipitating extracellular calcium , which prevents
the loss of calcium homeostasis. Because the mechanisms of action of hypothermia,
alkalinization and adenosine differ, when combined they can produce a greater degree of
neuroprotection than any one alone. Experiments are underway to collect more data to allow
statistical analysis of the observed influences, to determine the specific mechanisms by which
these techniques provide neuroprotection, and to determine whether they provide
neuroprotection against other excitotoxins and oxidative stressors. Although the mechanism s of
neuroprotection are not established, the data suggest that under selective conditions, the clinical
perfusion of brain and spinal cord post trauma will provide neuroprotection.

Trauma and stress lead to neurotoxicity and neurological deficits. Therefore, post trauma, rapid
interventions are required to provide neuroprotection and prevent neuron death and the
consequent loss of neurological functions. Although many interventions have been tested in
different injury models, none alone provides sufficient neuroprotection, although combined
approaches provide enhanced neuroprotection and improved maintenance of neurological
integrity. But, further advances are required to find neuroprotective conditions that provide
enhanced neuron viability and maintenance of neurological function following trauma. One
challenge to finding a good combination of neuroprotective approaches is that trauma causes a
cascade of events whereby a number of them simultaneously contribute to neurotoxicity.
Therefore, an ideal neuroprotective technique requires the application of metho            unters
the varied neurotoxic mechanism triggered by trauma.

Electrophysiological and other studies on adult rat DRG neurons have shown that small changes
in cellular environment (predominantly increases in temperature and decreases in pH), result in
dramatic changes in membrane properties that result in neuron death [1]. However, trauma-
induced excitotoxicity and oxidative stress induce massive neurotoxicity via large changes in
intracellular and extracellular pH, temperature changes due to inflammation, acidification, and
loss of calcium homeostasis. An important question is how the neurotoxic actions of these
changes can be inhibited to maintain neuron viability.

This paper focuses primarily on neurotoxicity induced by excitotoxicity and oxidative stress and
several approaches to providing neuroprotection . Although the initial mechanisms of action of
excitotoxicity and oxidative in initiating neurotoxicity differ, both excitoxicity and oxidative
stress contribute to similar, and in some cases identical, underlying mechanisms of neurotoxicity.
Thus, neuroprotective techniques that are effective against one will provide protection against
the other, and when combined the influences will be complimentary.

DRG neurons contain and release excitatory amino acids (EAAs), such as glutamate and
aspartate [2-11] and they have receptors for these EAAs [6, 12-15]. Ischemia-induced secondary
causes of neurotoxicity [16], include lipid peroxidation [17], trauma-induced inflammation [18,
19], and excessive release of excitatory amino acids (EAAs) (excitotoxins) [4-7, 10, 20-25].
Exposure to an excessive concentration of EAAs leads to the excessive stimulation of the N-
methyl-D-aspartic acid (NMDA) subtype of glutamate receptors and the development of a
neurotoxic level of extracellular acidification (pH 7.3 to 6.5), with the extent of neuron death
related to the degree and duration of acidosis [6, 26]. Both NMDA receptor activation and
acidification result in the massive influx of extracellular calcium [5], which disrupts neuron calcium
homeostasis causing neuron death [27-29]. The excessive release of glutamate also stimulates
further glutamate release in a positive feedback loop by interacting with non -NMDA receptor
subtypes [6], which open N-type voltage-sensitive calcium channels thus allowing further
excessive entry of calcium into neurons causing loss of calcium homeostasis and neuron death
[30]. Simultaneously, calcium-induced calcium release, accompanied by a further influx of calcium
through voltage-gated calcium channels after glutamate-induced depolarization, contributes to
glutamate-induced toxicity.

Oxidative stress involves depletion of mitochondrial glutathione (mtGSH) and increased
hydrogen peroxide (H2 O2 ) generation [31]. Cyanide induces oxidative stress as a rapidly acting
mitochondrial poison that inhibits cellular respiration and energy metabolism (glycolysis)
leading to histotoxic hypoxia followed by cell death. Cyanid e toxicity is accompanied by lipid
peroxidation, reactive oxygen species (ROS) and reactive nitrogen species (RNS) , and
diminished cellular antioxidant status. These cascading events triggered apoptotic neuron death
[32]. Cyanide is an inhibitor of the antioxidant system and mitochondrial function and involves
both reactive oxygen species and nitric oxide-mediated oxidative stress as an initiator of
apoptosis [33].

Acidosis is a universal tissue response to oxidative stress induced by ischemia and is caused by
cyanide due to its triggering a calcium -dependent massive release of EAA transmitters. The
acidosis resulting from the accumulation of extracellu EAAs is linked to worsening cerebral
infarction, in part from the restoration of oxidative metabolism following the oxidative stress and
from neurotoxicity via EAA activation of NMDA-receptors [34]. Neuroprotection against
cyanide can be provided by lowering of the extracellular pH following cyanide- induced acidosis,
by suppressing pH-sensitive mechanisms of injury, and by inhibiting glycolysis (blocking
oxidative metabolism ) with 2-deoxyglucose, which substitutes for glucose but cannot be
metabolized. Thus sodium entry via is prevented by blocking the Na+/H+ exchanger. Similarly
neuroprotection can be retarded by blocking NMDA receptor activation [35, 36] . These latter
mechanisms of action are supported by the finding that MK-801, the NMDA receptor antagonist,
significantly reduces neuron death when administered following ischemia -induced metabolic
inhibition [36].

Hypothermia also decreases glucose and oxygen metabolic rates while maintaining a slightly
better energy level. This indicates that ATP breakdown is reduced more than its synthesis,
leading to improved neuron survival, which means that secondary failures in energy
requirements are prevented.[37]

For adult rat dorsal root ganglion (DRG) neurons, 400 µM -4 mM of cyanide causes sustained
increases in intracellular calcium which leads to apoptosis, with the extent of neuron death
increasing with time after exposure. Therefore the response of neurons to cyanide response
provides a simple assay for testing neuroprotective agents [38].

Excitotoxic- and oxidative stress-induced neurotoxicity are linked because both are associated
with neuron exposure to excess glutamate. This in turn causes a large increase in intracellular
calc ium, although the cyanide-induced calcium increase is greater than that induced by glutamate
because cyanide also induces the release of calcium from intracellular pools [39]. The cyanide
response is mimicked when oxidative metabolism is disrupted by sodium azide, oligomycin, or
dinitrophenol [39].

Short exposure of DRG neurons to hypoxia results in a reversible elevation of intracellular
calcium, which can be almost completely eliminated by removal of extracellular calcium sodium,
or the external application of nifedipine, an L-type calcium channel blocker [40]. Thus the
hypoxia-induced elevation of cytosolic calcium is induced by combined changes of function of

voltage-operated L-type calcium and sodium channels and calcium accumulation by

Noncompetitive NMDA receptor antagonists and enzymatic glutamate degradation abolish both
glutamate- and cyanide-induced calcium increases [39]. The disruption of calcium homeostasis is
reduced by reducing extracellular calcium. These results suggest that extracellular glutamate
accumulation and activation of glutamate receptors are involved in the cyanide neurotoxicity
response. However, during metabolic compromise, glutamate-induced calcium elevations are
enormously amplified, and in spite of cyanide inducing large increases in intracellular calcium
concentrations, cyanide neurotoxicity is not as extensive as that of glutamate, indicating that a
general elevation in cytoplasmic calcium does not necessarily predict neurodegeneration [39].
On the other hand, the finding that cyanide induces a 32% increase
calcium levels [41] supports the hypothesis that calcium plays an important role in cyanide-
mediated neurotoxicity, although the magnitude of the initial intracellular calcium concentration
change does not predict the toxicity of an agonist on NMDA receptors [42].

Extracellular alkalinization results in intracellular alkalinization [43]. Intracellular alkalinization
stimulates glycolysis while independently enhancing energy generation [37]. Therefore, both
hypothermia and alkalinization would be anticipated to counter the neurotoxic influences of 2-

Neurotoxic oxygen free radical production
Ischemia induces the generation of neurotoxic levels nitric oxide (NO) free radicals which are
associated with intracellular acidification. This acidification can be prevented by intracellular
alkalinization to pH 7.4 during NO exposure [44]. Therefore, alkalinization may provide
neuroprotection by preventing extracellular acidification and/or by reducing the production of
oxygen free radicals.

Lipid peroxidation
Cyanide is neurotoxic by inducing hydroperoxide generation and subsequent lipid peroxidation
which causes membrane structure and functional damage [45]. The importance of lipid
peroxidation in cyanide-induced neurotoxicity is indicated the fact that blocking lipid
peroxidation provides neuroprotection against cyanide [46].

Neuron lipid -derived radical formation is pH dependent, with decreasing extracellular pH
causing more neuronal toxicity [47]. Therefore, alkalinization should provide neuroprotection
against cyanide poisoning by reducing lipid peroxidation.

NMDA receptor antagonists
Neuroprotection against excessive activation of NMDA receptors is provided by competitive
antagonists of the NMDA receptors such as MK-801 or 6 -cyano-7-nitroquinoxaline-2,3 -dione
(CNQX), and alpha-amino-3 -hydroxy -5-methylisoxazole-4-propionic acid/kainate (AMPA/KA)
[48 -5 0]. Similarly, in a dose-dependent manner, bradykinin (0.001 to 1 µM) is neuroprotective
against glutamate-induced neurotoxicity [51].

Whole body and localized hypothermia provide neuroprotection to adult CNS neurons against
infarct- induced ischemia [52 -54], and to spinal cord neurons against compromised blood flow
and reperfusion [55-59] . Part of this neuroprotection results from the expression of heat shock
proteins [60, 61]. Hypothermia to 33o C [62] reduces NMDA channel activation , thereby reducing
NMDA receptor-mediated excitatory postsynaptic potential amplitude [63], which in turn
reduces the number of EAA-activated NMDA receptors, while simultaneously shortening the
activated channel open time. Reducing NMDA receptor activation also reduces calcium influx
thereby preventing the disruption of calcium homeostasis. The neuroprotection provided by
hypothermia is enhanced when it is combined with simultaneous infusion of NMDA receptor
antagonists [64] and is increased 23-fold when combined with alkalinization [65, 66].

Alkalinization provides neuroprotection against ischemia of adult rat CNS and adult human DRG
neurons [53, 67, 68] and against azide-induced chemical anoxia [69]. Alkalinization to pH 8.2
prevents the development of acidosis induced neurotoxicity against azide-induced chemical anoxia
[69], although it is not known whether it acts by preventing EAA induced acidosis, or blocking the
actions of glutamate [70].

At a normal extracellular calcium concentration, activation of large numbers of neuronal calcium
channels leads to massive entry of extracellular calcium ions, causing excessive intracellular
calcium accumulation, loss of calcium homeostasis and neurotoxicity. The relative concentration
of extracellular calcium is pH dependent, with calcium requiring a pH of less than 6 to enter
solution. At physiological pH (7.6) calcium solubility is 160 mg/l, and increases to 6,390 mg/l at
pH 7.0, but decreasing 40-fold to 10.1 at pH 8.4 [71]. With increasing extracellular pH, most
calcium is precipitated into calcium bicarbonate, which reducing the concentration of soluble
extracellular available to enter neurons. Therefore, alkalinization provides neuroprotection
against calcium toxicity.

Since cyanide causes a rapid decrease in intracellular pH which would increase calcium
solubility , alkalinization in the presence of cyanide would reduce the free calcium available to
enter neurons making increased pH neuroprotective against cyanide-increased free extracellular
calcium [72]. This is supported by the finding that pretreating neurons with a calcium channel
blocker delays reduces the magnitude of the intracellu pH increase thereby reducing cyanide
toxicity [72] . Similarly, removing intracellular calcium by chelation with BAPTA partially
increases the cyanide-induced neurotoxicity [73].

Adenosine is a nucleoside that is rapidly formed by neurons and glial cells in large amounts
during ischemia due to the intracellular breakdown of adenosine triphosphate (ATP). The
adenosine is transported into the extracellular space where it provides endogenous
neuroprotection by counteracting the generation of the neurotoxic increases in extracellular
calcium concentration [74-76].

Adenosine acts through three subtypes of receptors, A1, A2A and A3A, located on neurons, glial
cells, blood vessels, platelets, and leukocytes, and which are linked via G-proteins to different

effector systems, such as adenylate cyclase and membrane ion channels. A1 receptor agonists,
A2A agonists and antagonists, and A3A R antagonists, protect against a range of insults, both in
vitro and in vivo [77, 78] . Both acute and chronic treatments with these ligands can produce
diametrically opposite outcome effects, probably due to adaptational changes in receptor number
or properties. By activating the A2A receptor, adenosine provides neuroprotection by causing the
release of neurotrophic factors and cytokines, such as interleukins and tumor necrosis factor-
alpha from immune-competent leukocytes and glia [79, 80] . Adenosine A2A receptor activation
also promotes transmitter release and postsynaptic depolarization. Selective A2A receptor
antagonists protect against neurons from death caused by ischemia, excitotoxicity and free
radicals [77]. They also decrease neuronal loss induced by excitatory amino acids (EAA) [81]
and provide protection against neurodegenerative processes such as Parkinson's disease [82]. In
most animal models, adenosine A2A receptor antagonism attenuates inflammation through
control of the proliferation and production of proinflammatory cytokines [79, 83]. A2A receptors
also modulate some general cellular processes to affect neuronal cells death by fine tuning
neuronal and glial functions to produce neuroprotective effects [84] . Acting via A2A receptors,
adenosine improves cerebral microcirculation thereby providing oxygen and a substrate supply
to tissues [74, 82].

Adenosine A1 receptor agonists, or inhibitors of cellular reuptake and inactivation of adenosine,
provide neuroprotection against glutamate-induced excitotoxicity by blocking calcium influx
mediated by the NMDA receptor and preventing the loss of intracellular calcium homeostasis
[81]. A1 adenosine receptor activation suppresses neural activity by a predominantly presynaptic
action [77] probably by directly stabilizing the neuronal membrane potential by increasing the
conductance for potassium and chloride ions. This blocks glutamate induction of an uncontrolled
membrane depolarization via ion channel-linked glutamate receptors of the NMDA type [85].
This activation triggers pathological blocking of voltage-sensitive potassium currents, increasing
NMDA receptor-mediated calcium influx, and impairing glutamate uptake by astrocytes [74, 86].
Adenosine provides neuroprotection by suppressing the urotoxic GABA-activated current
(IGADA) in a majority of neurons [59].

Neuron dissociation
Dorsal root ganglia (DRG) were removed at room temperature (20o C) from euthanized adult
male Sprague Dawley rats and placed in DMEM + F15 (50/50) (Sigma Chemical, St. Louis)
culture medium without serum and treated as previously described [67]. Briefly, the DRG
connective tissue capsules were removed using irredectomy scissors and the DRG cut into small
pieces and the pieces of DRG placed in culture medium containing collagenase P (5 mg/ml),
neutral dispase II (8 mg/ml), and DNase (0.3 mg/ml) (Roche Diagnostics, Indianapolis, IN) and
then placed in an O2 /CO2 incubator at 37o C for 1½ hour. Every 15 minutes the DRG pieces were
gently triturated 5 times through a 1-ml plastic pipette tip with an inside diameter just larger than
the pieces of DRG. The DRG neurons were completely dissociated in about 1 hour and 20
minutes. The enzyme-containing medium was replaced with normal medium at the 20o C and pH

Culture dishes
A circle with a diameter of about 20 mm was drawn on the bottom of the Falcon tissue culture
dishes, using a felt pen, and the area within the circ e treated for 1 hour with poly- l-lysine (1
mg/ml) (Sigma Chemical), washed, and then treated for 1 hour with laminin (Sigma Chemical)
(5 mg/ml), washed and 3 mm of normal culture medium added to each dish. Using a
micropipette, with a tip just larger than the neurons, attached to a mouth sucker, the dissociated
neurons were plated in the culture dishes within the c le drawn on the dish bottoms. Live
neurons immediately attached to the bottom of the culture dishes.

One hour after plating the neurons the culture medium was then replaced with medium at 36o C at
pH 7.6 or at 20o C and pH 9.3 containing the potassium cyanide glutamate, or potassium cyanide
plus 2-deoxyglucose. The temperature of the medium was maintained at 36o C by placing one set
of dishes in a CO2 incubator, while the other set of dishes was placed on a cooling platform set to
20o C. The dishes were maintained under their appropriate temperature and pH for 15 , 3 0 and 45
and 60 minutes after which the medium was changed for medium at 20o C and pH 7.6, Followed
by placing the neurons in the CO2 incubator at 36o C.

Stabilizing the pH of the culture medium
The culture medium was adjusted to pH 9.3 using HEPES buffer and sodium hydroxide. Adding
glutamate to the culture medium made it acidic. Therefore, for studies of glutamate toxicity,
glutamate was added to the culture medium and the medium then adjusted of the appropriate pH
with sodium hydroxide.

Neuron viability assay
Immediately after treating the cultures with glutamate or potassium cyanide, the yield of viable
neurons was assessed using the Trypan blue dye exclusion test. Trypan blue was added to the
cultures to a final concentration of 0.4%. After 20 minutes the number of viable neurons within
the circle on the bottom of each dish was counted under bright field optics. These numbers were
used as the starting valued for the number of viable neurons in each dish against which to
compare the number of viable neurons after each dish had been exposed to glutamate or
potassium cyanide under normal conditions of temperature and pH or hypothermic and alkaline

After 18 hours the number of viable neurons in each culture dish was counted.

Control adult rat DRG neurons were exposed only to normal tissue culture medium without any
additional factors at physiological temperature (36o C) and pH (7.6) after being plated. Eighteen
hours after plating the neurons, the yield of viable neurons was 88%.

After the neurons had been plated the influences of glutamate and potassium cyanide on neuron
viability was determined by rapidly raising glutamate or potassium cyanide to the culture
medium to a final concentration of 10 mM for 15, 30, 45 and 60 minutes. After the appropriate
exposure time, the glutamate and cyanide were removed by substituting the medium with normal

medium. As soon as the glutamate was added to the cultures the medium became acidic,
indicated by the change in color of the pH indicator dye in the culture medium. No pH changes
were observed upon addition of potassium cyanide.

Eighteen hours after exposing neurons to 15 minutes of glutamate, only 1.5% of the neurons
remained alive, based on Trypan blue dye exclusion analysis, and no viable neurons survived 30
minute exposure to glutamate.

Eighteen hours after exposing neurons to 15 minutes of potassium cyanide, only 2.0% of the
neurons remained alive, based on dye exclusion analysis, and no viable neurons survived 30
minute exposure to glutamate.

Protection by hypothermia and alkalinization
To determine whether hypothermia (20o C) and alkalinization (pH 9.3) provided neuroprotection
against the glutamate and cyanide toxicity seen in the previous experiments, both glutamate (10
mM) and potassium cyanide (10 mM), were added to the medium for 15, 30, 45 and 60 minutes,
after which they were washed out.

By 18 hours after exposure to glutamate for 15, 20, 45 and 60 minutes, the yield of viable
neurons was 85, 80, 78 and 75% respectively. The number of viable neurons after 15 minutes of
glutamate exposure represents a 57-fold increase compared to that of neurons in control cultures.

When exposed to potassium cyanide (500 µM ), for 15, 30, 45 and 60 minutes, the yield of viable
neurons maintained in hypothermic (20o C) and alkaline (pH 9.3) culture medium decreased to
65, 60, 53 and 50% respectively. The number of viable neurons after 15 minutes of glutamate
exposure represents a 22-fold increase compared to that of neurons in control cultures.

Neuroprotection by adenosine
An experiment was performed to determine whether the neuroprotection against potassium
cyanide provided by hypothermia (20o C) and alkalinization (pH 9.3) would be influenced by the
presence of adenosine (1 mM). Dissociated cultured neurons were therefore exposed to
potassium cyanide (10 mM) for 15, 30, 45 and 60 minutes while the neurons were maintained
hypothermic and alkaline culture medium. Eighteen hours after potassium cyanide exposure,
neuron viability was 87, 85, 80 and 77% respectively.


                                                                   control (36oC / pH 7.6)l

              % viable neurons pre- vs. post-exposure
                                                                                               cyanide + adenosine (20 C / pH 9.3)
                                                                                                glutamte (20 C / pH 9.3)
                                                                   cyanide (20oC / pH 9.3)



                                                                   cyanide (36oC / pH 7.6)
                                                                   glutamate (36 C / pH 7.6)

                                                              10        20             30           40            50            60
                                                                                     time of exposure (minutes)

Figure 1.
Plot of the % yield of viable adult rat DRG neurons immediately exposed for 15 to 60 minutes to
glutamate and cyanide under hypothermic and alkaline conditions vs. the yield of v      neurons
after 18 hours. The data show that hypothermia and alkalinization each provide neuroprotection
against glutamate and oxidative stress increasing neuro viability by 64 and 90% respectively, and
that the neuroprotection provided by hypothermia and alkalinization against cyanide is enhanced
when combined with simultaneous treatment with adenosine.

Experiments were performed on dissociated adult rat DR G neurons to determine whether
hypothermia and alkalinization provides neuroprotection against the excitotoxic neurotransmitter
glutamate, and the oxidative stressor cyanide. Control DRG neurons were exposed to glutamate and
cyanide (both at 10 mM) for 15 to 60 minutes under physiological conditions of temperature (36o C)
and pH (7.6), while experimental neurons were exposed to glutamate and cyanide under
hypothermia (20o C) and alkaline (pH 9.3) conditions. Following exposure to glutamate and cyanide,
the culture medium in all dishes was changed to physiological conditions of temperature and pH
(37o C and pH 7.6). Because glutamate- and cyanide-induced neuron death increases with time after
exposure, neuron viability was analyzed one hour after exposure to glutamate and cyanide and then
again 18 hours later to determine the number of surviving neurons.

Eighteen hours after a 15 minute exposure to glutamate and cyanide under physiological
temperature and pH, the number of viable neurons was 1.5 and 2% respectively . No neurons
survived more than 30 minutes of exposure to glutamate and cyanide. However, when neurons were
exposed to glutamate or cyanide for 15 minutes under hypothermic (20oC) and alkaline (pH 9.3)
there was a 57- and 33-fold, respectively, larger yield of viable neurons. Further, under hypothermic
and alkaline conditions, neurons survived up to 60 min tes of exposure to glutamate and cyanide,
with the yield of viable neurons surviving 60 minutes of exposure being respectively 50 and 77% of
the initial number of viable neurons.

The 50% lethal dose of glutamate for cerebral neurons is 10 µM [87]. In the present experiments the
neurons were exposed to 10 mM glutamate, a 1000-fold greater concentration, which under
physiological conditions of temperature and pH killed 98.5% of the neurons. However, when
exposed to this concentration of glutamate under hypothermic and alkaline conditions 85% of the
neurons survived. This indicates the potent degree of neuroprotection provided by hypothermia and

DRG neurons were also exposed to cyanide for 15, 30, 45 and 60 minutes to adenosine (1 mM) to
determine whether it enhanced neuroprotection provided by hypothermic and alkalinization .
Adenosine increased the yield of viable neurons by 25, 29, 34 and 35% respectively compared that
than provided by hypothermia and alkalinization alone. Thus, the neuroprotective influence of
adenosine was additive to those of hypothermia and alkalinization.

These data raise the question of the mechanisms by which glutamate and cyanide neurotoxicity was
caused , and how hypothermia and alkalinization might have provided neuroprotection.

Glutamate excitoxicity: excessive NMDA receptor stimulation
Neuron exposure to excessive amounts of an extracellular excitatory amino acid (EAA), such as
glutamate, causes excessive and neurotoxic activation of neuron NMDA receptors [6]. This is in
part due to the opening of N-type voltage -sensitive calcium channels, which allow excessive
entry of calcium into neurons [5], which causes the loss of calcium homeostasis, which leads to
neuron death [28-30]. However, the excessive rise in intracellular calcium also induces DRG
neurons to release the EAAs they contain, such as glutamate and aspartate [2-11], and because the
same neurons that release these EAAs possess receptors for them [6], the EAA release causes
neuronal excitation leading to a vicious cycle of further toxic stimulation and neuro death.
Therefore, neuron death due to glutamate exposure in the present experiments when the neurons
were maintained under physiological temperature and pH could have resulted from excessive
stimulation of the neuron NMDA receptors and the loss of calcium homeostasis.

Neurotoxicity due to glutamate and cyanide acidification
Excessive amounts of extracellular glutamate cause extracellular acidification . In the present
study addition of 10 mM glutamate shifted the culture medium from pH 7.6 to 6.5. Earlier
experiments showed that the extent of neuron death is related to the degree and duration of
extracellular acidosis [26 ]. However, the observed neuron death could also be attributed to
extracellular acidification .

Acidification and alkalinization causing loss and stabilization of intracellular calcium
Anoxia leads to an initial neuronal intracellular acidific ation, followed by a subsequent increase
in intracellular pH and further alkalinization after the return to normoxia [88, 89] . Cyanide-
induced anoxia also causes acidosis and a decreased intracellular pH [90]. These changes are due
to increased Na+ /H+ exchange activity involving hydrogen ion extrusion and Na+ /H+ exchange
[88]. Inhibiting Na+ /H+ exchangers prevents alkalinization [89]. Therefore, anoxia-induced
activation of a Na+ /H+ exchanger is involved in the pH shift [89].

Cyanide causes the excessive influx of calcium into isolated adult rat DRG neurons [91], which
could cause neuron death. The anoxia- induced increase in intracellular calcium is significantly
inhibited in nominally calcium -free medium. Changing the extracellular pH to 8.2 or 6.6, or the
addition of NMDA or non-NMDA receptor antagonists (D-AP5 and CNQX) in combination,
significantly reduce the increase in intracellular calcium, indicating a neuroprotective effect of
both extracellular alkalinosis and acidosis on the neurotoxic roles of excitatory amino acids [89].
Similarly, the loss of calcium homeostasis due to excess influx of calcium , and the release of
calcium from intracellular stores, can be blocked by the use of intracellular calcium chelators

Chick DRG neuron N-type calcium channel currents are extremely sensitive to small changes in
intracellular pH in the range produced by physiological and pathological events which produce
intracellular pH changes of 0.15 and 0.5 units. Between pH 7.4 and 6.8, N-type calcium
channels, which are intimately involved in ex ocytosis and other excitable cell processes, are
sensitive to intracellular pH changes [92]. Intracellular acidification from pH 7.3 to 6.6
reversibly inhibits calcium currents, while alkalinization from pH 7.3 to 7.5 potentiates calcium
currents [92] . Even in the absence of intracellular calcium, removed by a calcium chelator,
calcium channels are sensitive to intracellular pH changes. Therefore, some influences of
intracellular pH do not involve a calcium -dependent mechanism and the modulation of N-type
calcium channels. Further, intracellular pH may play an important role in physiological processes
that produce small changes in internal pH, and may play an important neuroprotective role in
pathological mechanisms producing larger intracellular pH changes.

Mechanisms of neuroprotection by hypothermia and alkalinization
These preliminary data were derived from pilot experiments aimed at seeing whether there are
indications that hypothermia and alkalinization provide neuroprotection against hypothermia and
alkalinization. Therefore it was not the intention of these experiments to determine the cellular
mechanisms by which neuroprotection might be provided. However, the preliminary data allow
us to speculate on the mechanisms by which they protection is provided by hypothermia and

Changing extracellular pH of DRG neurons results in a similar change in their intracellular pH
[93, 94]. Therefore, it should be possible to prevent the neurotoxicity caused by glutamate-
induced extracellular acidification by extracellular alkalinization.

Increasing neuronal extracellular pH from 7.4 to 8.5 causes a dramatic increase in the time (126
to 216 seconds) required for neurons to recover from a glutamate (3 µM, for 15 seconds)-
induced increase in intracellular calcium concentration , and removing extracellular calcium does
not block this prolonged recovery [94] . Since extracellular alkalinization causes rapid
intracellular alkalinization following glutamate exposure, this suggests that extracellular
alkalinization delays calcium recovery by affecting the intra-neuronal calcium buffering
mechanisms, rather than by an exclusively extracellular effect. The effect of extracellular
alkalinization to pH 8.5 calcium recovery is completely inhibited by the mitochondrial Na+ /Ca+
exchange inhibitor CGP-37157, suggesting that increased mitochondrial calcium efflux via the
mitochondrial Na+ /Ca+ exchanger is responsible for the prolongation of intracellular calcium
recovery caused by alkaline pH following glutamate exposure [94].

Decreasing extracellular pH induces a corresponding decrease in intracellular pH [95].
Acidification inhibits low external calcium -induced neuronal excitation by inhibiting the activity
of the csNSC channels [95]. Both the extracellular and the intracellular sites are involved in the
proton modulation of the calcium -sensing non-selective cation (csNSC) channels. Decreasing
extracellular calcium concentrations activate slow and sustained inward currents through the
csNSC channels [95]. Intracellular alkalinization mimics the potentiation of the csNSC, while
intracellular acidification by addition and subsequent withdrawal of NH4Cl mimics the
inhibition of the csNSC currents by decreasing extracellular pH [95]. Thus, acidification may
inhibit low external calcium-induced neuronal excitation by inhibiting the activity of the csNSC
channels, with both extracellular and the intracellula ites being involved in the proton
modulation of the csNSC channels.

In a nominally calcium -free medium, the initial acidification is followed by a significant
alkalinization. At an external pH of 8.2, the alkalinization is significantly increased, while at an
external pH of 6.2, the initial acidification is followed by further acidification in about half the
neurons [89]. Therefore, because intracellular pH follows extracel lar pH the, acidification
induced by oxidative stress should be blocked by external alkalinization.

Intracellular alkaline shifts stimulate calcium release from intracellular stores [96]. The internal
calcium increase is significantly reduced by removing external calcium [96]. Extracellular
alkalinization induces an increase of intracellular ca ium via the combined actions of
intracellular calcium -accumulating structures, the endoplasmic reticulum and mitochondria [97].
Since external alkalinization to pH 9.3 eliminates free extracellular calcium, it should prevent an
increase in intracellular calcium originating from extracellular sources.

Since both glutamate and cyanide induce extracellular acidification, decreased intracellular pH,
the influx of extracellular calcium and the release of calcium from intracellular stores, both
glutamate and cyanide can induce neurotoxicity by causing the loss of calcium homeostasis.
Therefore, alkalinization of the dissociated rat DRG neurons in the present experiments should
block both glutamate- and cyanide-induced intracellular acidification and loss of calcium
homeostasis thereby providing neuroprotection.

Alkalinization protects against NMDA receptor activation
NMDA receptors are pH sensitive with an alkaline pH shift to pH 8.7 reducing their activation
[98]. Alkalinization to pH 8.2 prevents the death of mouse neocortical neurons in primary culture
against azide-induced chemical anoxia and the resulting development of acidosis [69].

The present data are consistent with these data and in     that alkalinization provides
neuroprotection against glutamate-induced acidification. However, they do not discriminate
between whether the neuroprotection was provided by acidification, excitotoxicity or both.
Neuroprotection against excessive activation of NMDA receptors can be provided by
competitive antagonists of the NMDA receptors [48-50]. Therefore, if neuroprotection is
provided by the NMDA receptor blocker MK-801 in the presence of glutamate, this would
indicate that neuroprotection was by preventing excessive NMDA, and not by preventing
acidification .

Neuroprotection by hypothermia
Mild h ypothermia to 33-35o C is neuroprotective by blocking excessive activation of NMDA
receptors and neuron death [62, 99]. Therefore, the neuroprotection against glutamate
neurotoxicity in the present study that was provided by hypothermia may have been due to
blocking NMDA receptor activation, reducing the number of EAA-activated NMDA receptors,
while simultaneously shortening the channel open time of the activated channels. Both of these
actions would reduce calcium influx and prevent the disruption of calcium homeostasis.

Reducing free radical production
Ischemia- and cyanide-induced generation of nitric oxide (NO) free radicals oduces neurotoxicity.
The toxic effects of NO -triggered intracellular acidification can be prevented by intracellular
alkalinization to pH 7.4 during the initial 30 min of NO exposure [44]. In the present experiments,
extracellular alkalinization to pH 9.3 may have induced intracellular alkalinization that provided
neuroprotection and /or by reducing the production of oxygen free radicals.

Neuroprotection against prolonged ischemia is enhanced when hypothermia [57, 100, 101] and
alkalinization [69, 70] are each combined with adenosine, or when hypothermia and
alkalinization are combined [101]. The present data show that when all three are combined
neuroprotection is even greater than that provided by co       hypothermia and alkalinization.

In agreement with previous data [57, 100] the present data show that hypothermia and
alkalinization provide neuroprotection to adult rat DRG neurons exposed to potassium cyanide,
azide and glutamate compared to neurons exposed to the         pounds at normal temperature
and pH.

Examination of the influences of excitotoxicity and oxidative stress show that they induce
neuron death by both separate as well as related mechanisms and that several different
techniques can inhibit their neurotoxicity allowing adult neurons to survive excitotoxicity and
oxidative stress. We have examined several potential approaches for providing neuroprotection
and preliminary data show that hypothermia and alkalinization combined provide
complementary neuroprotection against glutamate and oxidative stress neurotoxicity because
their neuroprotection acts in slightly different, but overlapping ways. We also show that the
neuroprotection against cyanide that is provided by simultaneous hypothermia and alkalinization
can be enhanced when combined with adenosine. Therefore, we hypothesize that localized
venous perfusion of a region of brain or spinal cord that has undergone trauma with hypothermic
and alkaline saline, especially when combined with adenosine, will provide potent
neuroprotection and prevent neurological losses.

These studies were carried out in accordance with NIH Guide for the care and use of laboratory
animals, and under an IACUC-approved animal study proposal. All efforts were made to
minimize the number of animals used, and their suffering. This research was performed under an
IACUC-approved animal study proposal. No external funding was used to support this research.

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