Neurons in the Heterotopia Raddy L. Ramos1, Akm Rahman2 1

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Lack of hippocampal VEGF and VEGFR2 expression in the hypoxia tolerant
naked mole rat
Dan McCloskey1, Sharry Goldman2, Bruce Goldman2
1Psychology
and Neuroscience, College of Staten Island/CUNY; 2Ecology and Evolutionary Biology, Univ. of Connecticut, Storrs.
INTRODUCTION
40
FIG. 1. Naked mole rats housed in a series of cages connected by polycarbonate tubing.
Animals are housed according to colony with up to 50 animals cohabitating. Connections
are nearly air-tight allowing for manipulation of oxygen levels in the housing system. All
animals are implanted with RFID tags and movement through readers spaced throughout
housing system is measured. Photo by Jonathan Peters.
*
30
25
20
15
10
FIG. 3. VEGF expression levels in the hippocampi and lung of mice, rats, and naked mole rats under
different treatment conditions (see table below for details). Voluntary exercise elevated hippocampal
VEGF in mice, and hypoxia-induced seizures elevated hippocampal VEGF in rats, but naked mole rat
hippocampal VEGF levels were undetectable regardless of age, oxygen levels or seizures. Naked mole
rat lung tissue was included as a positive control to confirm specificity of the ELISA antibody for naked
mole rat VEGF. * Significant t-test result when compared to same species control.
FIG. 4. Immunocytochemical labeling of the neuron specific
marker (green) and the VEGFR2 receptor (red) showed a
low level of VEGFR2 expression in the hippocampus, which
was primarily restricted to epithelium. This low level of
expression provides supporting evidence that VEGF
expression is unusually low in the naked mole rat
hippocampus.
Treatment Conditions
Species
Mouse
Rat
IMMUNOCYTOCHEMISTRY:
To determine the presence of VEGFR2 naked mole rats were anesthetized with
urethane (1.25 mg/kg) and perfusion fixed through the aorta with 4%
paraformaldehyde. The brains were removed and sectioned at 50mm using a
vibratome. Free floating sections were washed in 0.1 m a series of Tris buffers (pH
7.6), some containing 10% triton X or 0.05% bovine serum albumin. Sections were
then incubated in 1:5000 Alexa 488 conjugated mouse anti-NeuN (Millipore) and
1:1000 rabbit anti- VEGFR2 (KDR, FLK-1; Upstate) overnight at room temperature.
After more buffer rinses, sections were incubated in anti-rabbit Dylight 549 secondary
antibody (Pierce), washed and coverslipped. Sections were imaged on a Leica SP-1
Laser scanning confocal microscope.
*
Naked Mole Rat
0
TREATMENTS:
See Table.
VEGF ELISA:
Samples were added in duplicate to a Calbiochem Anti-mouse VEGF Sandwich
ELISA kit (EMD Biosciences), according to the manufacturer’s instructions at a final
concentration of 15% protein. The assay was analyzed on a plate reader at 450nm
and quantified. Samples were compared against a serial diluted mouse VEGF
standard, and values of non standard blank wells were subtracted from the final optical
density readings.
Rat
5
METHODS
TISSUE PROCESSING:
Animals were deeply anesthetized with CO2 and decapitated. Brains were
removed and colled rapidly using ice cold sucrose based artificial cerebrospinal fluid.
Hippocampi were isolated and frozen in isopentane cooled to -40°C on dry ice. Lung
tissue was extracted and frozen in the same manner. Samples were stored at -80°C
for up to two months.
Tissue samples were weighed and homogenized manually in a protein inhibitor
lysis buffer containing NP-40, EDTA, Apronitinin, Leupeptin, and Pepstatin. Tissue
composed 30% of the total homogenate solution. Homogenate suspensions were
centrifuged for 30 minutes at 13,000G (4°C) and supernatants were collected.
Mouse
35
VEGF Protein (pg/mg tissue)
The naked mole rat (Heterocephalus glaber) is one of 18 species of African
mole-rats, which belong to the hystricognath suborder of rodents. Of all African molerats, the naked mole rat has the harshest environmental habitat in the arid regions of
central and eastern Ethiopia, central Somalia, and Kenya. They are exclusively
subterranean and maintain an extensive burrow system, with 3-4 km of tunnels, in
order to identify tuber food sources.
During the rainy seasons there is substantial ground saturation and flooding of
the burrow system which greatly limits the penetration of oxygen from the ground
surface. Field measures of mole rat burrows estimate that these animals live in
prolonged periods of hypoxia, with O2 levels as low as 7% (compared to 21% in air,
Shams et al. 2005).
Several pieces of evidence suggest that NMRs have adapted physiologically to
carry out normal functions in a low oxygen environment. First studies of blood oxygen
level show that NMRs have a higher level of blood oxygenation than rats and mice
(Johansen et al. 1976). Second, other species of African mole rats have been shown to
have elevated Hif1α and erythropoetin levels in blood (Shams et al. 2004). Third,
excised hippocampal tissue of naked mole rats has been shown to withstand oxygen
deprivation for longer periods than rats or mice (Larson & Park, 2009; Nathaniel et al.
2009). Fourth, metabolic rates are lower than animals of comparable size and circadian
rythms are not environmentally controlled (Riccio and Goldman, 2000). Together, these
findings suggest that this species has adapted at the systemic and local brain level to
withstand hypoxia.
VEGF is an oxygen-regulated cytokine secreted by multiple cell types in the
brain and periphery. A major contributor to its expression is hypoxia, which results in
the binding of hypoxia-inducible factors (HIFs) to the hypoxia response element (HRE)
located in the promoter region of the VEGF gene (Carmeliet et al., 1998; Marti & Risau,
1998). The ability of HIFs to upregulate VEGF under hypoxic conditions is likely related
to the potent mitogenic effect that VEGF exerts on vascular endothelial cells to
increase angiogenesis and vascular permeability to increase the amount of oxygen and
nutrients delivered to metabolically-compromised neurons (Ruiz de Almodovar et al.
2009). However, it is now known that VEGF exerts direct effects on neurons that may
promote survival and suppress neuronal activity under hypoxic conditions (McCloskey
et al. 2005; 2009).
The purpose of the current study was to compare the level of expression of
VEGF in the naked mole rat hippocampus to traditional rodent species exposed to
manipulations likely to upregulate hippocampal VEGF expression. The results indicate
that hippocampal VEGF expression is significantly lower in the hippocampus of the
naked mole rat than it is in rats or mice. This may serve as an additional piece of
evidence that the naked mole rat brain is specially equipped to tolerate a large
variation in oxygen availability.
RESULTS
Naked Mole Rat
FIG. 2. Naked mole rats maintained at 3% CO2 (10x higher
than air) for 96 hours did not appear to have diminished
activity levels in the colony. CO2 levels were regulated by a
programmable Biospherix oxygen sensor and valve.
References
Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell
P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E (1998) Role of HIF-1alpha in hypoxia-mediated
apoptosis, cell proliferation and tumour angiogenesis. Nature 394:485-490.
Johansen K, Lykkeboe G, Weber RE, Maloiy GM (1976) Blood respiratory properties in the naked mole rat heterocephalus
glaber, a mammal of low body temperature. Respir Physiol 28:303-314.
Larson J, Park TJ (2009) Extreme hypoxia tolerance of naked mole-rat brain. Neuroreport 20:1634-1637.
Marti HH, Risau W (1998) Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor
and its receptors. Proc Natl Acad Sci U S A 95:15809-15814.
McCloskey DP, Croll SD, Scharfman HE (2005) Depression of synaptic transmission by vascular endothelial growth factor
in adult rat hippocampus and evidence for increased efficacy after chronic seizures. J Neurosci 25:8889-8897.
McCloskey DP, Hintz TM, Scharfman HE (2008) Modulation of vascular endothelial growth factor (VEGF) expression in
motor neurons and its electrophysiological effects. Brain Res Bull 76:36-44.
Nathaniel TI, Saras A, Umesiri FE, Olajuyigbe F (2009) Tolerance to oxygen nutrient deprivation in the hippocampal slices
of the naked mole rats. J Integr Neurosci 8:123-136.
Riccio AP, Goldman BD (2000) Circadian rhythms of body temperature and metabolic rate in naked mole-rats. Physiol
Behav 71:15–22
Ruiz de Almodovar C, Lambrechts D, Mazzone M, Carmeliet P (2009) Role and therapeutic potential of VEGF in the
nervous system. Physiol. Rev. 89: 607-648
Shams I, Avivi A, Nevo E (2004) Hypoxic stress tolerance of the blind subterranean mole rat: Expression of erythropoietin
and hypoxia-inducible factor 1 alpha. Proc Natl Acad Sci U S A 101:9698-9703.
Shams I, Avivi A, Nevo E (2005) Oxygen and carbon dioxide fluctuations in burrows of subterranean blind mole rats
indicate tolerance to hypoxic-hypercapnic stresses. Comp Biochem Physiol A Mol Integr Physiol 142:376-382.
Treatment
Control
Voluntary Exercise
Control
Perinatal Hypoxia
n
4
4
3
4
10 Day Old
Control
Hypoxia
Status Epilepticus
Lung Tissue
2
6
3
2
8
Description
Male and female 4 month old CD-1 mice were housed individually in standard tub cages with access to locked running wheels for 14 days.
Male and female 4 month old CD-1 mice were housed individually in standard tub cages with access to freely rotating running wheels for 14 days.
Male and female Sprague Dawley rats were maintained in a chamber for 30 minutes at normal oxygen on P10 and were sacrificed 24 hours later.
Male and female Sprague Dawley rats (P10) were maintained in a chamber infused with N2 gas at 6% O2 for 12 minutes followed by an O2 reduction
at 1%/min until the onset of apnea. Animals were sacrificed 24 hours later.
Male naked mole rats were sacrificed 10 days after birth.
Adult male and female (1-3 years old) naked mole rats from two separate colonies were sacrificed.
Adult male and females were sacrificed after 96 hours in hypercapnic hypoxia (3% CO2/ 18% O2) in the home cage.
Adult male naked mole rats were sacrificed 2-5 hours after prolonged seizures induced by 380 mg/kg pilocarpine hydrochloride)
Adult male and females were sacrificed under control, hypoxia, or status epilepticus conditions (pooled data)
CONCLUSIONS
1. Two weeks of voluntary exercise increases hippocampal VEGF expression in the adult mouse.
2. Hypoxia-induced seizures significantly increase VEGF expression in the hippocampus of the newborn rat.
3. Naked mole rats have comparatively very low levels of VEGF expression in the hippocampus under baseline conditions and
following hypoxia, seizures or birth.
4. Low levels of VEGFR2 expression suggests that VEGF signaling does not play a prominent role in normal hippocampal function in
the naked mole rat hippocampus.
Acknowledgements
This work was supported by a PSC CUNY award. We thank
the animal care staff at the College of Staten Island. We also
thank Chuanhua Wang for technical assistance and Drs. Bill
L’Amoreaux and Jeff Goodman for advice.
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