Potentially neuroprotective gene modulation in an in vitro model of mild traumatic brain injury

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Molecular and Cellular BiochemistryAn International Journal for ChemicalBiology in Health and Disease ISSN 0300-8177 Mol Cell BiochemDOI 10.1007/s11010-012-1541-2

Potentially neuroprotective genemodulation in an in vitro model of mildtraumatic brain injury

Valentina Di Pietro, Angela M. Amorini,Barbara Tavazzi, David A. Hovda,Stefano Signoretti, Christopher C. Giza,Giacomo Lazzarino, et al.

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Potentially neuroprotective gene modulation in an in vitromodel of mild traumatic brain injury

Valentina Di Pietro • Angela M. Amorini • Barbara Tavazzi • David A. Hovda •

Stefano Signoretti • Christopher C. Giza • Giacomo Lazzarino • Roberto Vagnozzi •

Giuseppe Lazzarino • Antonio Belli

Received: 10 September 2012 / Accepted: 6 December 2012

� Springer Science+Business Media New York 2012

Abstract In this study, we investigated the hypothesis

that mild traumatic brain injury (mTBI) triggers a con-

trolled gene program as an adaptive response finalized to

neuroprotection, similar to that found in hibernators and in

ischemic preconditioning. A stretch injury device was used

to produce an equi-biaxial strain field in rat organotypic

hippocampal slice cultures at a specified Lagrangian strain

of 10 % and a constant strain rate of 20 s-1. After 24 h

from injury, propidium iodide staining, HPLC analysis of

metabolites and microarray analysis of cDNA were per-

formed to evaluate cell viability, cell energy state and gene

expression, respectively. Compared to control cultures,

10 % stretch injured cultures showed no change in viabil-

ity, but demonstrated a hypometabolic state (decreased

ATP, ATP/ADP, and nicotinic coenzymes) and a peculiar

pattern of gene modulation. The latter was characterized by

downregulation of genes encoding for proteins of com-

plexes I, III, and IV of the mitochondrial electron transport

chain and of ATP synthase; downregulation of transcrip-

tional and translational genes; downregulation and upreg-

ulation of genes controlling the synthesis of glutamate and

GABA receptors, upregulation of calmodulin and cal-

modulin-binding proteins; proper modulation of genes

encoding for proapoptotic and antiapoptotic proteins.

These results support the hypothesis that, following mTBI,

a hibernation-type response is activated in non-hibernating

species. Unlike in hibernators and ischemic precondition-

ing, this adaptive gene programme, aimed at achieving

maximal neuroprotection, is not triggered by decrease in

oxygen availability. It seems rather activated to avoid

V. Di Pietro � A. Belli

Neuropharmacology and Neurobiology Section, School of

Clinical and Experimental Medicine, College of Medical and

Dental Sciences, University of Birmingham, Birmingham, UK

V. Di Pietro

Division of Clinical Neurosciences, School of Medicine,

University of Southampton, Southampton, UK

A. M. Amorini � B. Tavazzi � G. Lazzarino

Institute of Biochemistry and Clinical Biochemistry, Catholic

University of Rome, Rome, Italy

D. A. Hovda

Department of Neurosurgery, Department of Molecular and

Medical Pharmacology, David Geffen School of Medicine at

UCLA, UCLA Brain Injury Research Center,

Los Angeles, CA, USA

S. Signoretti

Division of Neurosurgery, Department of Neurosciences

Head and Neck Surgery, S. Camillo Hospital, Rome, Italy

C. C. Giza

Division of Pediatric Neurology, Department of Neurosurgery,

David Geffen School of Medicine at UCLA, Mattel Children’s

Hospital UCLA, UCLA Brain Injury Research Center,

Los Angeles, CA, USA

R. Vagnozzi

Division of Neurotraumatology and Neuroradiology, Department

of Biomedicine and Prevention, University of Rome Tor

Vergata, Rome, Italy

G. Lazzarino (&)

Division of Biochemistry and Molecular Biology, Department

of Biology, Geology and Environmental Sciences, University

of Catania, Viale A. Doria 6, 95125 Catania, Italy

e-mail: [email protected]

123

Mol Cell Biochem

DOI 10.1007/s11010-012-1541-2

Author's personal copy

increase in oxidative/nitrosative stress and apoptosis during

a transient period of mitochondrial malfunctioning.

Keywords Mild traumatic brain injury �Gene expression � Mitochondrial dysfunction �Hypometabolism � Hibernation � Neuroprotection

Introduction

Traumatic brain injury (TBI) is the leading cause of death

and disability under 45 years of age [1] accounting for

34 % of all deaths from trauma [2]. Advances in the

understanding of the mechanisms of TBI-induced neuronal

cell death underpin modern critical care management of the

severely injured brain [3]. However, mild TBI (mTBI) has

received far less attention and remains poorly understood

[4, 5], and it has been named the silent epidemic [6, 7].

It has been shown that mTBI leads to a transient state of

‘‘metabolic vulnerability’’ [8, 9] characterized by sudden

changes in ionic fluxes [10], release of excitatory amino

acids [11], altered rate of glucose metabolism [12], mito-

chondrial dysfunction [13–15], imbalance in energy

metabolism [16–18], and N-acetylaspartate (NAA) homeo-

stasis [19–21]. During this window of brain vulnerability, a

second mTBI may have the same dramatic consequences

observed after a severe TBI [9, 18]. Differential gene

expression following graded TBI has been demonstrated in a

rat model of lateral fluid percussion [22, 23] and in a model of

organotypic hippocampal slice cultures (OHSC) [24],

strongly suggesting that changes in gene expression are

strictly related to injury severity. In mTBI, characterized by

minimal or absent permanent histopathological damage, this

gene modulation may be interpreted as an adaptive strategy

to achieve neuroprotection. This strategy would require an

optimization of the metabolic rate during the transient met-

abolic depression following mTBI. If this was the case, it

should be possible to evidence clear molecular signs of

controlled hypometabolism in post-injured cerebral cells.

An example of hypometabolism in nature is hibernation, a

behavioral, physiological, and molecular adaptation to with-

stand protracted periods or seasons of insufficient or unpre-

dictable food availability, during which gene modulation

aimed to obtain neuroprotection has been demonstrated [25].

Similar to hibernation, ischemic preconditioning is another

example of adaptive hypometabolism. Through selective

changes in the gene expression [26] it increases tolerance to

sustained, long-term ischemia in the myocardium [27–29] and

it decreases ischemic brain damage caused by prolonged

middle cerebral artery occlusion, if preceded by brief, tran-

sient ischemic period [30].

Using a microarray approach, we hypothesized that a

stretch injury equivalent to mTBI would modulate gene

expression of specific metabolic pathways and cell func-

tions to achieve transitory hypometabolism and consequent

energy conservation. Similarities and differences between

mTBI and hibernation, aimed at evaluating the existence of

a cell program of neuroprotection based on gene modula-

tion, are presented.

Materials and methods

Preparation of OHSC and stretch induction

The OHSC were prepared using a method initially descri-

bed by Morrison et al. [31] and all procedures used were in

accordance with UK regulations under the Animals (Sci-

entific Procedures) Act of 1986.

Briefly, hippocampi from 8- to 10-day-old male Wistar

rats were isolated and sliced at a thickness of 400 lm,

using a McIlwain tissue chopper (Harvard Apparatus,

Edenbridge, UK). The slices were then placed on a silicone

membrane (0.25 mm; Specialty Manufacturing, Saginaw,

MI) attached to a custom-made stainless steel well, which

was previously treated with a coating solution of 320 lg/

mL of poly-D-lysine (Sigma) and 80 lg/mL of laminin

(Sigma) in sterile distilled water. The cultures were fed

initially in 1,200-lL NeuroBasal-A medium (Invitrogen,

UK), containing B27 supplement (Invitrogen) (1-mL/100-

mL medium), 5-mg/mL glucose (Sigma), 1-mM glutamine

(Sigma) and incubated at 37 �C in 95 % O2 ? 5 % CO2.

After 2 days in vitro, the NeuroBasal-A medium was

replaced with full serum-containing medium, consisting of

25 % heat-inactivated horse serum, 25 % Hanks’-balanced

salt solution, 50 % minimum essential medium (all Invit-

rogen), 1-mM glutamine (Sigma), and 5-mg/mL D-glucose

(Sigma). After approximately 10 days, when cultures

adhered to the membrane and showed clear definition of

the neuronal regions-CA1, CA3 and dentate gyrus [31], the

medium was removed from the healthy cultures and the

wells were clamped to the stretch injury device. The

stainless steel well and silicone membrane were then dis-

placed over a fixed, hollow indenter to generate an equi-

biaxial strain field, subjecting the cultures to a single

stretch injury at a specified Lagrangian strain of 10 % and a

constant strain rate of 20 s-1, known to induce cell injury

with no decrease in viability, thus being comparable to

mTBI. The strain was controlled by a linear actuator (BEI

Kimco), and maintained under feedback control through a

linear encoder (Renishaw, UK) and motion control board

(Precision Motion Dynamics, Canada). Forty-five OHSC

were prepared. Fifteen of them were incubated for 24 h at

37 �C in 95 % O2 ? 5 % CO2 and used as controls. Of the

30 OHSC receiving a 10 % stretch injury, half were

incubated for 24 h and half for 72 h, under the same

Mol Cell Biochem

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conditions of controls. At the end of incubations, both five

controls and ten 10 % stretched OHSC (of these, five

incubated for 24 and five for 72 h) were used to measure

viability. The remaining 10 OHSC of each group (controls,

10 % stretched at 24 and 72 h) were pooled and divided

into two aliquots: one was used to carry out the gene

expression analysis and the other was used to carry out the

HPLC assay of metabolites. The above reported experi-

ment was repeated three times for a total of 45 control

OHSC and 90 10 % stretched OHSC.

Propidium iodide (PI) fluorescence and cDNA

microarray

At the desired time point, 5 lg/mL of PI (Molecular

Probes, Paisley, UK) was added to the medium for 2 h; the

cultures were then examined under a Leica DM-IRBE

epifluorescence microscope (Leica Microsystems Ltd.,

UK) to assess the percentage of cell death. Images were

acquired using a 5XNA 0.12 lens and a cooled Hamamatsu

camera, and analyzed with Volocity (Improvision, UK)

3-D imaging software. The mean percentage area of PI

fluorescence for controls and the two 10 % stretched

OHSC were analyzed and compared.

Total RNA was extracted from tissue cultures in a mon-

ophasic solution of phenol and guanidine isothiocyanate

(Trizol; Invitrogen) as previously described [18]. The

integrity and concentration of the total RNA were deter-

mined using the RNA 6000 Nano Assay Kit and a Bioana-

lyzer 2100 according to the manufacturer’s protocols

(Agilent, UK). The labelling of complementary RNA

(cRNA) with the fluorophores cyanine-3 (cy-3) and cyanine-

5 (cy-5) (PerkinElmer/NEN Life Sciences, UK) and its

subsequent amplification was completed using the Low

RNA Input Linear Amplification Kit (Agilent) according to

the manufacturer’s protocol. The cRNA was assessed using a

NanoDrop ND-3300 Fluorospectrometer (Agilent) to ensure

that sufficient cRNA of appropriate quality had been pre-

pared. Once labelling was completed, equal amounts of cy-3-

and cy-5-labeled probes were simultaneously applied to a

4X44 K whole rat genome microarray (Design 14879; Ag-

ilent) for a competitive hybridization reaction using a gene

expression hybridization kit (Agilent), according to the

manufacturer’s protocol. Following hybridization, the array

was washed using the gene expression wash buffer kit (Ag-

ilent), dried and then scanned using a DNA microarray

scanner (Agilent). The microarray datasets, preprocessed by

Agilent Feature Extraction software, were analyzed by

Genespring GX 10.0.2. Automatic flags applied by the

Feature Extraction software were used to localize and

excluded from the analysis if any probe that was non-uni-

form, population outliers, saturated, absent, not positive, and

significant, or not above background. The signal from each

spot was calculated as the average intensity minus the

average local background. Expression ratios of cy-5:cy-3

were normalized using LOESS, a method that takes into

account and corrects for intensity-dependent artefacts in the

measurements. The mean log2 ratios for each probe were

found across the two replicates, and the probes that revealed a

[1.5-fold difference in expression (higher or lower) com-

pared to the control sample were identified. Genes of interest

were divided into six categories (Tables 1, 2, 3, 4, 5, 6).

Real-time-quantitative polymerase chain reaction

(RT-qPCR)

Also with the aim to confirm the microarray data, all genes

presented in Table 1 (Ndufa11, ND1, ND2, ND5, CYTB,

COX2, COX3, ATP5J) have been analyzed in RT-qPCR

either in control or in 10 % stretched cells at 24- and 72-h

post-injury. Initially, the reverse transcription of total RNA

was performed for all samples. Subsequently, 1 lg of total

RNA, 500 ng of oligo dT primers (Roche Molecular Bio-

chemicals, UK), and 200 U of Superscript II Reverse

Table 1 ETC genes

Gene

symbol

Gene name Transcript ID Gene

regulation

Fold

change

Ndufa11 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 11 NM_212517 Down -2.09

ND1 NADH dehydrogenase subunit 1 TC615875 Down -2.42

ND2 NADH dehydrogenase subunit 2 ENSRNOT00000040993 Down -2.18

ND5 NADH dehydrogenase subunit 5 ENSRNOT00000048767 Down -2.09

CYTB Cytochrome B, complex III ENSRNOT00000042098 Down -2.11

COX2 Cytochrome oxidase C, subunit 2 ENSRNOT00000043693 Down -2.14

COX3 Cytochrome oxidase, subunit 3 ENSRNOT00000049683 Down -2.12

ATP5J ATP synthase, H? transporting, mitochondrial F0 complex, subunit

F6

NM_053602 Down -2.11

Gene expression analysis of ETC genes (complexes I, III, IV, and V), 24 h after mTBI. Genespring GX 10.0.2 was set to cut-off the changes in

the range ±1.5-fold

Mol Cell Biochem

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Table 2 Transcription genes

Gene symbol Gene name Transcript ID Gene

regulation

Fold

change

Dmrta2 Doublesex and mab3-related transcription factor like family 2 XM_233363 Down -2.26

Klf7 Kruppel-like factor 7 XM_343581 Down -2.54

Tcf12 Transcription factor 12 NM_013176 Down -2.88

Tsc22d3 TSC22 domain family, member 3 NM_031345 Up 2.55

RGD1565584 CREB/ATF bZIP transcription factor ENSRNOT00000025653 Down -2.28

LOC679869 Similar to transcription factor 7 like 2, variant 2 XM_001054844 Down -2.11

Tcfdp2 Transcription factor Dp2 XM_217232 Down -2.67

Tcf15 Transcription factor 15 XM_230722 Down -3.38

Atf6 Cyclic-AMP-dependent transcription factor ATF-6 alpha XM_222871 Down -2.43

Zfp91 Transcription factor activity attending zinc finger protein ENSRNOT00000016804 Down -2.31

TC642341 Transcriptional factor SOX1 TC642341 Down -2.85

Zfp36l2 Zinc finger protein 36 C3H type like 2 NM_001036626 Down -2.54

Nkx2-4 NK2 transcription factor related locus 4 XM_345445 Down -3.02

Pitx3 Paired-like homeodomain transcription factor 3 NM_019257 Down -2.23

Foxe3 Forkhead-related protein transcription factor 8 ENSRNOT00000010208 Down -3.14

Med25 Mediator of RNA polymerase II transcription subunit 25 XM_214950 Down -2.21

Zbtb7b Zinc finger BTB domain containing 7B XM_215633 Down -2.15

Snapc1 Small nuclear RNA activating complex, polypeptide 1 XM_234299 Up 2.12

RGD1563478 RNA polymerase II transcriptional coactivator XR_008667 Down -2.17

Surb7 Suppressor of RNA polymerase B homolog XM_232531 Down -2.13

Abca2 ATP-binding cassette subfamily A member 2 NM_024396 Down -2.15

Lancl2 LanC L antibiotic synthetase component C-like 2 ENSRNOT00000008948 Up 2.38

Cbfa2t1 CBFA2T1 identified gene homolog XM_342808 Up 2.34

Trib3 Tribbles homolog 3 NM_144755 Up 2.35

Vps36 Vascular protein sorting-associated protein 36 ENSRNOT00000016995 Down -2.20

Foxe1 Forkhead box E1 NM_138909 Down -2.70

Nr4a3 Neuron-derived orphan receptor 1 DQ268830 Up 2.20

Jund Jun D proto-oncogene NM_138875 Down -3.03

Egr1 Early growth response 1 NM_012551 Up 2.40

Cnot7 CCR4-NOT transcription complex subunit 7 XM_224894 Up 2.19

Bhlhb2 Basic helix loop helix domain containing class B2 NM_053328 Up 2.13

NKx6-2 Similar to homeotic protein Gtx XM_219447 Down -2.31

Zfp131 Zinc finger protein 131 ENSRNOT00000021484 Down -2.53

Tcea1 Transcription elongation factor A (SII)1 NM_001025735 Down -2.36

Nr4a3 Nuclear receptor subfamily 4, group A, member 3 variant 1 NM_031628 Down -2.26

Hmgb2 High mobility group box 2 BC089854 Down -2.22

Crabp2 Cellular retinoic acid binding protein 2 NM_017244 Down -2.90

Klf5 Kruppel-like factor 5 NM_053394 Down -2.18

Nfix Nuclear factor 1/X NM_030866 Down -2.36

Rbpsuh Recombining protein suppressor of hairless XM_232595 Down -2.64

Smad7 SMAD homolog 7 NM_030858 Down -2.12

Klf10 Kruppel-like factor 10 NM_031135 Up 2.10

Mxd1 Max dimerization protein 1 ENSRNOT00000023856 Down -3.21

RGD1560268 Zinc finger homeobox 3 ENSRNOT00000019408 Down -2.55

Atrx Alpha thalassemia/mental retardation syndrome X-linked ENSRNOT00000033355 Down -2.20


Nfkbiz Nuclear factor of kappa light polypeptide gene enhancer in B cells

inhibitor

XM_221537 Up 2.26

Mol Cell Biochem

123


Transcriptase in a total volume of 20 lL of 1 9 first-strand

buffer (Invitrogen) were incubated at 42 �C for 60 min.

The concentration and purity of the resulting cDNA was

determined with a ND-1000 UV–Vis Spectrophotometer

(NanoDrop). RT-qPCR was carried out on the single-

stranded cDNA yielded from the reverse transcription

using a one-step PCR kit, Rat Custom RT-qPCR assay for

use with SYBR green chemistry (PrimerDesign Ltd.,

Southampton, UK), and a Bio-Rad iCycler iQ5 PCR

Thermal Cycler (Bio-Rad, Hercules, CA, USA) according

to the manufacturer’s protocol, for 40 cycles. The primers

for the genes selected for RT-qPCR were designed using

Primer 3 Input software (primer3_www.cgi v.0.2). For

accurate gene expression measurements with RTqPCR, the

constitutively expressed housekeeping gene hypoxanthine

phosphoribosyltransferase 1 (HPRT 1, Rattus norvegicus,

NM_012583.2) was selected after verifying that there was

no relevant differences between control and treated sam-

ples. At the end of reactions, melting curves were gener-

ated to check the specificity of PCR reactions. The relative

levels of mRNA expression for each gene in each sample

were calculated according to the 2-DDCt method.

OHSC processing and HPLC analysis of metabolites

Cultures (controls and 10 % stretched after 24 or 72 h) were

deproteinized using an organic solvent deproteinization pro-

cedure previously reported [32]. Each protein-free sample was

filtered through a 0.45-lm HV Millipore filter (Millipore

Corp., Billerica, MA) and loaded (20 lL) onto a Hypersil

C-18, 250 9 4.6 mm, 5-lm particle size column, provided

with its own guard column (Thermo Fisher Scientific, Milan,

Italy) and connected to a high-performance liquid chroma-

tography (HPLC) apparatus consisting of a SpectraSystem

Table 2 continued

Gene symbol Gene name Transcript ID Gene

regulation

Fold

change

Foxc2 Forkhead box C2 XM_001079064 Down -3.38

Pou3f4 Pou domain, class 3, transcription factor 4 NM_017252 Down -2.20

Scand1 SCAN domain containing 1 XM_342558 Down -2.19

Mnt Max-binding protein XM_220698 Down -2.39

Brd1 Bromodomain containing 1 XM_235552 Down -2.77

Prrx2 Paired-related homeobox 2 XM_238327 Down -3.19

Nr4a1 Nuclear receptor subfamily 4, group A, member 1 NM_024388 Up 2.47

Trak2 Trafficking protein, kinesin binding 2 NM_133560 Down -2.18

Fos FBJ murine osteosarcoma viral oncogene homolog NM_022197 Up 2.17

Hand1 Heart and neural crest derivatives expressed transcript 1 BC097327 Down -2.12


Zfhx1b Zinc finger E-box-binding homeobox 2 NM_001033701 Down -2.47

Cdkn1c Cyclin-dependent kinase inhibitor 1C(P57), variant 1 NM_001033757 Down -2.12

Cdkn1c Cyclin-dependent kinase inhibitor 1C(P57), variant 3 NM_182735 Down -2.50

LOC257642 rRNA promoter-binding protein transcription factor binding NM_147136 Down -2.64

Hist3h2a Histone cluster 3, H2a NM_021840 Down -2.34

H2a Histone H2A type 1-F ENSRNOT00000059342 Down -2.65

Hist3h2ba Histone cluster 3, H2ba XM_220506 Down -2.14

Hist1h2af Histone cluster 1, H2af NM_001024282 Down -2.34

Myst3 Histone acetyltransferase 3 ENSRNOT00000037389 Down -2.42

RGD1562724 Similar to H3 family XM_225322 Down -2.14

H3f3b Histone H3 family 3B BC086580 Down -2.12

Hbxap Histone binding XM_218939 Down -2.50

Hist1H2aa Histone cluster 1, H2aa NM_021839 Down -2.16

Hist1h2bm Histone cluster 1, H2bm XM_341530 Down -2.28

Hist2h2be Histone cluster 2, H2be XM_227459 Down -2.13

Hist1h4b Histone cluster 1, H4b NM_022686 Down -2.61

Gene expression analysis of genes involved in the transcription process (transcription factors and cofactors, activators of the RNA polymerase,

negative and positive regulator of transcription, rRNA promoter binding proteins and histone proteins), 24 h after mTBI. Genespring GX 10.0.2

was set to cut-off the changes in the range ±1.5-fold

Mol Cell Biochem

123


http://www.cgi

P2000 pump system (Thermo Fisher Scientific) and a highly

sensitive UV6000LP diode array detector (Thermo Fisher

Scientific) equipped with a 5-cm light path flow cell and setup

between 200- and 300-nm wavelength. Data acquisition and

analysis were performed using a personal computer and the

ChromQuest software package provided by the HPLC man-

ufacturer (Thermo Fisher Scientific).

Metabolites related to cell energy state and mitochon-

drial function (ATP, ADP, AMP, oxypurines, nucleosides,

nicotinic coenzymes) were separated, in a single chro-

matographic run, according to a gradient modification of an

existing ion-pairing HPLC method [33]. Assignment and

calculation of the compounds of interest in chromato-

graphic runs of cell extracts were carried out at 260-nm

wavelength by comparing retention times, absorption

spectra, and areas of peaks with those of peaks of chro-

matographic runs of freshly prepared ultrapure standard

mixtures with known concentrations.

Statistical analysis

In microarray analysis, a two-sample t test, corrected by

Benjamini–Yekutieli for multiple testing was used to

evaluate significant differences among groups. Differences

were considered to be statistically significant if p \ 0.05.

Comparison of metabolite concentrations in control and

10 % stretched cultures was carried out using two-tailed

t tests with Bonferroni correction for multiple comparisons.

Differences were considered to be statistically significant if

p \ 0.05.

Table 3 Translation genes

Gene symbol Gene name Transcript ID Gene regulation Fold change

Eif4g3 Eukaryotic translation initiation factor 4 gamma, 3 XM_216563 Down -3.61

Eif4g1 Eukaryotic translation initiation factor 4 gamma, 1 XM_213569 Down -2.24

Eif3s10 Eukaryotic translation initiation factor 3, subunit 10 NM_001047087 Down -2.65

Eif4g2 Eukaryotic translation initiation factor 4 gamma, 2 ENSRNOT00000023040 Down -2.78

Tcea1 Translation elongation factor A (SII) 1 NM_001025735 Down -2.36

LOC288019 Similar to eukaryotic translation elongation factor 1a1 XM_006893 Up 2.14

LOC315661 Eukaryotic translation elongation factor 1a2 XR_006926 Up 2.31

Rpl15 Ribosomal protein L15 M83679 Down -2.13

RGD1565566 Similar to 60S ribosomal protein L18a XM_574336 Down -2.82

Mrpl51 Mitochondrial ribosomal protein L51 XM_216269 Down -2.22

LOC682368 Ribosomal protein L28 XM_001061225 Down -2.16

RGD1565258 Similar to 40S ribosomal protein S17 XM_344408 Down -2.45

Rps20 Ribosomal protein S20 NM_001007603 Down -2.21

Mrpl11 Mitochondrial ribosomal protein L11 NM_001006973 Down -2.16

Fau Finkel-Biskis-Reilly murine sarcoma virus NM_001012739 Down -2.10

Gspt1 G1 to S phase transition 1 NM_001003978 Down -2.15

Gene expression analysis of genes involved in the translation process (initiation and elongation factors, structural constituents of ribosome), 24 h

after mTBI. Genespring GX 10.0.2 was set to cut-off the changes in the range ±1.5-fold

Table 4 Calcium-related (A) neuromodulation and (B) calmodulin and calmodulin-binding proteins


(A)

Gria2 Glutamate receptor, ionotropic, AMPA 2 NM_017261 Up 2.12

Gabra1 Gamma-aminobutyric acid (GABA) A receptor, alpha 1 NM_183326 Up 2.15

Grin2d Glutamate receptor, ionotropic, N-methyl D-aspartate 2D NM_022797 Down -3.29

(B)

Calm3 Calmodulin3-phosphorylase kinase, delta NM_012518 Up 2.16

Rgs4 Calmodulin binding- regulator of G protein signalling 4 NM_017214 Up 2.16

Gap43 Calmodulin binding- grow associated protein 43-axonal regeneration NM_017195 Up 2.09

(A) Gene expression analysis of genes involved in the neuromodulation process, 24 h after mTBI. Genespring GX 10.0.2 was set to cut-off the

changes in the range ±1.5-fold. (B) Gene expression analysis of calmodulin and calmodulin-binding genes, 24 h after mTBI. Genespring GX

10.0.2 was set to cut-off the changes in the range ±1.5-fold

Mol Cell Biochem

123


Results

Evaluation of cell viability following 10 % stretch

injury by PI fluorescence

Viability of the slice cultures was observed at 24 and 72 h

after 10 % stretch or 24-h incubation in the case of con-

trols. The percentage area of red fluorescence compared to

the total area of the hippocampal slice of each image was

later analyzed. Comparing the stretch groups with controls,

the 10 % stretched slices either at 24- or 72-h post-injury

did not show significant differences in cell death (mean cell

death 2.56 ± 0.38 % in controls; 3.07 ± 1.01 % in 10 %

stretched at 24 h, 4.22 ± 1.88 % in 10 % stretched at

72 h), as expected for this model and severity of mTBI.

Analysis of the cell energy state and mitochondrial-

related metabolism

As representative parameters to evaluate the cell energy

state, ATP and ADP were measured in controls and 10 %

stretched OHSC at 24- or 72-h post-injury (Fig. 1a). Sig-

nificant depletion in ATP and concomitant increase in ADP

were observed in injured cells at 24 h after stretch

(p \ 0.01 in comparison to control), while normalization of

both energy metabolism-related parameters were recorded

72 h after stretch (not significant respect to controls;

p \ 0.001 respect to 24 h). At 24-h post-injury, this change

in both metabolites was responsible for a 44 % decrease in

the ATP/ADP ratio, an index of mitochondrial phosphor-

ylating capacity, which was viceversa not different from

control OHSC after 72 h from stretch induction (Fig. 1b).

As a consequence of an imbalance in ATP production and

consumption, the 10 % stretched OHSC at 24 h showed a

significant increase in the concentrations of the dephos-

phorylated ATP catabolites oxypurines (hypoxanthine,

xanthine, and uric acid) and nucleosides (inosine and

adenosine), with respect either to the corresponding values

recorded in control OHSC or to the values observed after

72 h of recovery from injury (Fig. 2a). The same Fig. 2b

illustrates the effect of the stretch injury on the sum of

reduced and oxidized nicotinic coenzymes NADH ? -

NAD? and NADPH ? NADP?. A general decrease in the

concentration of the nicotinic coenzyme pool was observed

in 10 % stretch OHSC 24 h after stretch with respect to

either controls or OHSC after 72 h of recovery (p \ 0.01).

Table 5 ATP-consuming proteins


Tlk1 Tousled-like kinase 1 XM_242032 Down -2.20

Map3k3 Mitogen activated protein kinase kinase kinase 3 XM_221034 Down -2.68

Rock1 Rho-associated coiled-coil forming kinase 1 NM_031098 Down -2.72

Kif5b Kinesin family member 5B NM_057202 Down -2.14

BACS Bile acyl-CoA synthetase TC633900 Down -2.96

Map4k4 Mitogen-activated protein kinase kinase kinase kinase 4 XM_217381 Down -2.26

Atrx ATP-dependent helicase ATRX ENSRNOT00000033355 Down -2.20

Prkwnk1 Protein kinase, lysine deficient 1 NM_053794 Down -2.94

Stk4 Serine/threonine-protein kinase 4 XM_230833 Down -2.66

Zcwcc1 Zinc finger, CW-type with coiled-coil domain 1 XM_214072 Down -2.27

Uhmk1 U2AF homology motif (UHM) kinase 1 NM_017293 Down -2.12

Map3k1 Mitogen activated protein kinase kinase kinase 1 NM_053887 Down -2.10

Slk Serine/threonine kinase 2 NM_019349 Down -2.31

Abca2 ATP-binding cassette, sub-family A NM_024396 Down -2.15

Csnk1a1 Casein kinase 1, alpha 1 NM_053615 Down -2.11

Lars Leucyl-tRNA synthetase NM_001009637 Down -2.22

ATP6V1F protein ATPase, H ? transporting, lysosomal 14kD, V1 subunit F TC630287 Down -2.35

Dclk1 Doublecortin-like kinase 1 NM_053343 Up 2.11

Papss2 30-Phosphoadenosine 50-phosphosulfate synthase 2 XM_215288 Up 2.32

Zap70 Zeta-chain (TCR)-associated protein kinase 7 NM_001012002 Up 2.25

Ret Ret proto-oncogene NM_012643 Up 2.35

Map3k4 Mitogen-activated protein kinase kinase kinase 4 XM_217804 Up 2.11

Stk25 Serine/threonine kinase 25 NM_184049 Up 2.24

Gene expression analysis of ATP-consuming proteins (mainly protein kinases), 24 h after mTBI. Genespring GX 10.0.2 was set to cut-off the

changes in the range ±1.5-fold

Mol Cell Biochem

123


In summary, biochemical parameters related to the cell

energy metabolism and mitochondrial functions clearly

indicate a transient state of hypometabolism induced by a

stretch injury consistent with mTBI.

Changes in gene expression following stretch injury

Data of cDNA microarray of selected groups of genes are

given in Tables 1, 2, 3, 4, 5, 6. The expression profile of

genes encoding for protein subunits of the complexes I, III,

and IV of the electron transport chain (ETC) and of ATP

synthase (complex V) that were affected by stretch injury

at 24-h post-damage, are reported in Table 1. These results

show that the genes responsible for the synthesis of com-

plex I subunits (specifically, Ndufa11 synthesizing NADH

dehydrogenase-ubiquinone 1 alpha subcomplex 11, ND1

synthesizing NADH dehydrogenase subunit 1, ND2 syn-

thesizing NADH dehydrogenase subunit 2 and ND5 syn-

thesizing NADH dehydrogenase subunit 5), of complex III

subunits (specifically, CYTB synthesizing cytochrome B,

complex III), and of complex IV subunits (specifically,

COX2 synthesizing cytochrome oxidase C, subunit 2 and

COX3 synthesizing cytochrome oxidase C, subunit 3) were

downregulated in this model of mTBI. Additionally, the

gene ATP5J encoding for ATP synthase, H? transporting,

mitochondrial F0 complex, subunit F6, exhibited a similar

two-fold decrease in expression, thereby indicating not

only a reduction in the overall capacity to transfer electrons

in the ETC but also a decrease in the proton pumping

through complex V necessary for ATP biosynthesis.

Microarray data of genes involved in the regulation of

the transcriptional machinery that were affected by 10 %

stretch injury (comparable in damage to a mTBI) are

illustrated in Table 2. Results indicate that the expression

of 74 genes encoding for transcriptional factors and

cofactors, activators of RNA polymerase, rRNA promoter-

binding proteins and histone proteins, was differently

influenced by the 10 % stretch injury with respect to con-

trols. Among these 74 genes, 59 were downregulated and

only 15 were upregulated. It is worth noting that 4 of the

upregulated genes (Lancl2, Cbfa2t1, Trib3, Bhlhb2) are

negative regulators of transcription. Hence, the final result

is that 10 % stretched OHSC exhibited an overall inhibi-

tion of transcription processes.

The effect of the 10 % stretch injury on genes involved in

the translational process, encoding for initiation and elon-

gation factors and structural constituents of ribosome, are

summarized in Table 3. These results showed significant

Table 6 Apoptosis-related (A) proapoptotic genes and (B) antiapoptotic genes


(A)

Sh3glb1 SH3-domain GRB2-like B1 NM_001011929 Down -2.23

Rock1 Rho-associated coiled-coil forming kinase 1 NM_031098 Down -2.72

Stk4 Serine/threonine-protein kinase 4 XM_230833 Down -2.66

Tnfsf12 Tumor necrosis factor ligand superfamily member 12 NM_001001513 Down -2.21

Phlda1 Pleckstrin homology-like domain, family A, member 1 NM_017180 Down -2.17

Map3k1 Mitogen-activated protein kinase kinase kinase 1 NM_053887 Down -2.10

Klf10 Kruppel-like factor 10 NM_031135 Up 2.10

Ddit4 DNA-damage-inducible transcript 4 NM_080906 Up 2.18

Trib3 Tribbles homolog 3 NM_144755 Up 2.35

Nr4a1 Nuclear receptor subfamily 4, group A, member 1 NM_024388 Up 2.47

Vdac1 Voltage-dependent anion channel 1 NM_031353 Up 2.53

(B)

Ccl2 Chemokine (C–C motif) ligand 2 NM_031530 Up 2.48

Vegfa Vascular endothelial growth factor A NM_031836 Up 2.18

Birc3 Baculoviral IAP repeat-containing 3 NM_023987 Up 2.16

Tsc22d3 TSC22 domain family 3 NM_031345 Up 2.55

Bnip3 BCL2/adenovirus E1B 19-kDa interacting protein 3 NM_053420 Up 2.22

Son Son DNA binding protein ENSRNOT00000002769 Down -2.13

Gstp1 Glutathione-S-transferase, pi 1 NM_012577 Down -2.11

Gdnf Glial cell line derived neurotrophic factor NM_019139 Down -3.51

(A) Gene expression analysis of proapoptotic genes, 24 h after mTBI. Genespring GX 10.0.2 was set to cut-off the changes in the range ±1.5-

fold. (B) Gene expression analysis of antiapoptotic genes, 24 h after mTBI. Genespring GX 10.0.2 was set to cut-off the changes in the

range ±1.5-fold

Mol Cell Biochem

123


changes in the expression of 16 differentially regulated

genes, among which 14 were downregulated and only 2

upregulated.

Table 4 summarizes the effect of the 10 % stretch injury

on genes responsible for the synthesis of proteins involved

in neuromodulation (A) and of calmodulin and calmodulin-

binding proteins (B). The upregulation of the expression of

the alpha 1 GABA A receptor, of the AMPA2 subunit of

the ionotropic AMPA receptors, and the simultaneous

downregulation of the ionotropic, NMDA2D glutamate

receptor, indicate a complex response of OHSC injured

cells aimed to avoid excitoxicity caused by mTBI-induced

glutamate release and to counteract glutamate-mediated

Ca2? overloading. Such a gene strategy was also evident

by the upregulation of the genes encoding for calmodulin

and calmodulin-binding proteins (b). In particular, the

upregulation of Rgs4 had multiple effects, i.e., reducing the

glutamate signalling and inhibiting MAP kinase activity,

while the simultaneous upregulation of Calm3 and Gap 43

conferred an increased cell resistance to calcium-mediated

toxicity.

In Table 5, we report the change in expression of 23

genes responsible for the codification of ATP-consuming

proteins, 17 of which were downregulated and only 6 were

upregulated. Fourteen of these 23 genes encode for protein

kinases; out of these 14, 10 were downregulated, while the

remaining 4 were upregulated by the 10 % stretch injury. It

is of note that the downregulation of the Map3k1 gene and

upregulation of the Map3k4 gene, the protein products of

which (MEKK1 or mitogen-activated protein kinase kinase

1 and MEKK4 or mitogen-activated protein kinase kinase

4, respectively) are operative in the complex MAP kinase

pathway which regulates a conspicuous number of cellular

Fig. 2 Concentrations of oxypurines (hypoxanthine xanthine and uric

acid) and nucleosides (inosine, adenosine and guanosine) (a), deriving

from ATP catabolism, and values of nicotinic coenzymes (b) in

control and 10 % stretched organotypic hippocampal cell slices

(OHSC). Values are the mean of three experiments in which five

OHSC preparations were pooled either after 24-h incubation only

(controls), or after 10 % stretch injury and 24-h incubation (10 %

stretched). Standard deviations are represented by vertical bars.

Experimental conditions for OHSC preparation, stretch injury induc-

tion and HPLC conditions for metabolite determination are fully

described in ‘‘Materials and methods’’. *Significantly different from

controls (p \ 0.05)

Fig. 1 Concentrations of ATP and ADP (a) and values of the ATP/

ADP ratio (b), as a measure of the mitochondrial phosphorylating

capacity, in control and 10 % stretched OHSC. Values are the mean

of three experiments in which five OHSC preparations were pooled

either after 24-h incubation only (controls), or after 10 % stretch

injury and 24-h incubation (10 % stretched). Standard deviations are

represented by vertical bars. Experimental conditions for OHSC

preparation, stretch injury induction and HPLC conditions for

metabolite determination are fully described in ‘‘Materials and

methods’’. *Significantly different from controls (p \ 0.05)

Mol Cell Biochem

123


processes including activation of c-Jun and therefore

apoptosis. In general, it can be affirmed that one of the

responses of OHSC to mTBI is to decrease intracellular

signaling and, indirectly, to decrease ATP consumption.

The effects of the 10 % stretch injury on the expression

of proapoptotic and antiapoptotic genes, observed at 24-h

post-damage induction, are summarized in Table 6. Eleven

proapoptotic genes showed significant change in expres-

sion, 6 of which were downregulated and 5 were upregu-

lated (Table 6A), while 8 antiapoptotic genes underwent

significant modulation, 5 of which were upregulated and 3

downregulated (Table 6B). In accordance with our previ-

ous genontology results showing no induction of the genes

controlling the apoptotic process [24], the present results

indicate that the differential pattern in the expression of

proapoptotic and antiapoptotic genes leads to an overall

inhibition of apoptosis.

Real-time-quantitative polymerase chain reaction

To confirm the microarray data, as well as to verify whe-

ther changes in the expression of genes related to the

mitochondrial ETC were transitory and dependent on the

time interval from injury, we evaluated by RT-qPCR the

expression of Ndufa11, ND1, ND2, ND5, CYTB, COX2,

COX3, ATP5J in controls and 10 % stretched OHSC at 24

and 72 h from injury. As illustrated in Table 7, the RT-

qPCR data confirmed the decrease in expression of these

selected genes at 24-h post-injury, in perfect accordance

with what recorded with microarray (Table 1). Further-

more, measurement performed 72 h after the 10 % stretch

clearly indicates that expression of the ETC genes returned

to levels not different from those determined in controls.

Discussion

Transient mitochondrial malfunctioning following mTBI is

a well-established phenomenon [34, 35] leading to an

overall derangement of cell energy state and metabolism

(decrease in ATP, ATP/ADP ratio, creatine, creatine

phosphate, acetyl-CoA, N-acetylaspartate, etc.) [9, 17, 18,

36, 37]. In the present model of OHSC receiving a 10 %

stretch injury, we clearly found, at 24-h post-injury, a state

of hypometabolism connected to altered mitochondrial

function (decrease in ATP/ADP ratio and nicotinic coen-

zyme pool, increase in oxypurines and nucleosides; Figs. 1,

2). This was surprisingly accompanied (Table 1) by a

downregulation of genes encoding for different protein

subunits of complexes I, III, and IV of the ETC, as well as

of ATP synthase (complex V). This strongly supports

previous observations indicating that, following mTBI in

the rat, the imbalance in ATP homeostasis is due to a

decreased rate of mitochondrial use of oxygen for energy

production rather than to secondary hypoxic conditions

[16, 17]. On the other hand, it has been demonstrated that

continuation of mitochondrial oxidative phosphorylation in

the pathological milieu (i.e., during temporary mitochon-

drial malfunctioning) generates reactive oxygen species

[38], mitochondrial calcium overload [39], release of

cytochrome C [40]. In our experiments, the downregulation

of the ETC genes may have the role to decrease the rate of

electron transfer, thus decreasing the risks of triggering

oxidative/nitrosative stress during ETC malfunctioning

[38, 41]. Results obtained in 10 % stretched OHSC at 72-h

post-injury clearly show that mitochondrial functions were

only transiently altered as expression of the genes encoding

for different protein subunits of complexes I, III, and IV of

the ETC, as well as of ATP synthase (complex V), was not

different from that found in control OHSC (Table 7). A

significant confirmation of the transitory changes induced

by this type of damage was obtained by values of com-

pounds related to energy metabolism, the concentrations of

which at 72-h post-injury were not different from those

recorded in controls (Figs. 1, 2).

However, lowering the rate of ATP biosynthesis after

mTBI, when cells are engaged in re-establishing homeo-

stasis (ionic balance, oxidants/antioxidants balance, etc.)

and in reparative processes (membrane restoration,

re-folding of unfolded proteins, etc.) would be extremely

deleterious for the entire organ unless accompanied by a

concomitant ‘‘energy conserving program’’. According to

Tables 2, 3, and 5, the gene expression of proteins involved

in some relevant ATP-consuming processes in OHSC

(transcription, translation, and protein phosphorylation) is

drastically downregulated 24 h after a 10 % stretch injury,

Table 7 Expression of genes encoding for protein subunits of the

complexes I, III, IV, and V of the ETC (Table 1), as measured by RT-

qPCR in controls and 10 % stretched cells at 24- and 72-h post-injury

Control 24-h post-injury 72-h post-injury

Ndufa11/Hprt 1 ± 0.15 0.53 ± 0.12a,b 0.89 ± 0.21

ND1/Hprt 1 ± 0.08 0.62 ± 0.08a,b 1.36 ± 0.24

ND2/Hprt 1 ± 0.11 0.59 ± 0.15a,b 0.94 ± 0.14

ND5/Hprt 1 ± 0.03 0.51 ± 0.09a,b 1.08 ± 0.19

CYTB/Hprt 1 ± 0.12 0.68 ± 0.18a,b 1.11 ± 0.24

COX2/Hprt 1 ± 0.15 0.61 ± 0.23a,b 0.97 ± 0.07

COX3/Hprt 1 ± 0.12 0.70 ± 0.13a,b 0.98 ± 0.29

ATP5J/Hprt 1 ± 0.09 0.66 ± 0.18a,b 1.27 ± 0.28

The expression was calculated relatively to the housekeeping Hprt

gene

Values are the mean ± standard deviation of 3 preparations of 5

pooled OHSC eacha Significantly different from control; p \ 0.01b Significantly different from 72-h post-injury; p \ 0.01

Mol Cell Biochem

123


indicating a complex adaptive strategy aimed at optimizing

ATP consumption when ATP production is limited. This is

corroborated by data of Li et al. [23], indicating mild–

moderate injury causes downregulation of several gene

sets, while severe injury causes upregulation. It is therefore

conceivable that cells receiving milder injuries (and thus,

more likely to survive) have a molecular response designed

for energy recovery and repair, while those undergoing

severe injuries result in pathological injury cascades that

are not targeted to recovery.

In this light, the upregulation of ionotropic GABAA

receptors and AMPA2 (GluR2) subunit of the AMPA

receptor (Table 4A), should reduce calcium entry into the

cell and guard against excitotoxicity [42, 43], while the

downregulation of Grin2d encoding for the ionotropic

NMDA2D glutamate receptor (Table 4A) should counter-

act glutamate excitoxicity avoiding further calcium entry in

post-injured cells. A gene response aimed at restoring

calcium homeostasis is also evidenced by the simultaneous

upregulation of Calm3 and Gap43, both genes involved in

regulating intracellular calcium and to induce cell resis-

tance to calcium-mediated toxicity when overexpressed

[44].

The indication that the traumatized cerebral tissue acti-

vates a gene program as an adaptive response to afford

neuroprotection is corroborated by a similar pattern of gene

regulation physiologically occurring in hibernators. In win-

ter time, hibernators deal with two critical situations: limited

food supply and enormous energetic costs of maintaining a

constant high body temperature in cold weather [45].

Hibernating cells maintain viability by downregulating

oxygen consumption, ceasing non-essential cellular func-

tions and reducing ATP demand. Similar to what observed in

injured OHSC, it has been shown that the gene expression of

NADH-ubiquinone oxidoreductase and cytochrome C oxi-

dase [46–48] is downregulated in the brain of golden-man-

tled ground squirrels (Spermophilus lateralis) during late

torpor [49], a mechanism that affords them cell protection

[50]. Therefore, hibernators reorganize the priorities for ATP

use, controlling and coordinating the expression and the rate

of all ATP-generating and ATP-utilizing metabolic func-

tions [51–53]. As a consequence, some of the major ATP-

consuming processes, including transcription [54], transla-

tion [55], and ATP-dependent ion transport, are suppressed

during hibernation [56]. Even a striking inhibition of protein

phosphorylation in the brain of ground squirrel S. tride-

cemlineatus during hibernation has been observed [55],

thereby reinforcing a similarity between the pattern of gene

modulation of hibernators and that found in the post-injured

brain (downregulation of genes synthesizing different pro-

tein kinases, Table 5). This is even more evident when

considering that the general strategy of avoiding the induc-

tion of apoptosis recorded in our 10 % stretched OHSC

(Table 6) was observed in the greater horseshoe bat (Rhi-

nolophus ferrumequinum) [57].

Similarities and differences in the gene response

observed following mTBI and that occurring in hibernators

concern receptors for neuromodulators and modes for

controlling calcium overloading. Likewise to post-injured

OHSC (Table 4A), it has been shown that NMDA receptor

expression decreases during hibernation and increases upon

arousal [58], thereby contributing to afford protection from

persistent oxygen and nutrient deprivation [59]. Rather

than modulating the expression of neurotransmitter recep-

tors (Table 4A), hibernators downregulate calcium gating

channels [60] as they do not primarily face the post-mTBI

glutamate-mediated calcium overloading [10].

The causes of activation of the gene program of neu-

roprotection are likely to differ profoundly when consid-

ering ischemic preconditioning [27–30], hibernation and

this model of mTBI. In ischemic preconditioning and

hibernators, the common feature is the decreased oxygen

and substrate availability to tissues, fast in ischemic pre-

conditioning, slow in hibernators. As mitochondria are the

main target of this decrease in oxygen and substrate, it can

be assumed that modulation in gene expression is conse-

quent to protracted mitochondrial malfunction leading to

hypometabolism and progressive imbalance in ATP pro-

duction and consumption. In the case of the present model

of pure ‘‘in vitro’’ mTBI, we demonstrated that this gene

program of neuroprotection, similar to that of hibernators

and possibly of ischemic preconditioning, is not associated

with any change in substrates and oxygen during the whole

duration of the experiments, i.e., stretched and control

OHSC had full availability of substrates and oxygen at

constant concentrations. Also on the strength of our pre-

vious observations indicating sudden hypometabolism and

no differences in TBI damages between spontaneously

breathing and mechanically ventilated rats [13, 16], it can

be hypothesized that following mTBI mitochondria suffer

transient malfunctioning, specifically involving ETC and

its capacity to correctly handle the tetravalent reduction of

molecular oxygen, in spite of no change in oxygen and

substrate availability to cells. This decreases the amount of

ATP generation and might cause, if protracted, a dramatic

increase in oxidative/nitrosative stress. Hypometabolism

and, more importantly, signals derived from mitochondri-

ally produced oxygen and nitrogen radicals are likely to be

the factors triggering the gene program of neuroprotection,

having in the decrease of electron flow through the ETC

during transient mitochondrial malfunctioning, one of its

main effects/target (downregulation of complexes I, III,

and IV of the ETC; see Table 1). Therefore, the present

data demonstrate that following mTBI decrease in oxygen

and substrate availability is unnecessary to cause mito-

chondrial malfunction, hypometabolism and activate this

Mol Cell Biochem

123


program of neuroprotection. This also indicates that the

major molecular changes influencing the post-injury period

are triggered by the trauma itself and do not require sec-

ondary ischemic damage, i.e., eventual modifications in

cerebral blood flow following TBI are not the main

determinants for such molecular changes.

In conclusion, our data support the hypothesis that the

ability to depress temporarily the basal metabolic rate and

enter a hypometabolic or dormant state, through a targeted

modulation of gene expression, is a necessary strategy for the

traumatically injured brain. This adaptive physiological

mechanism may also explain why a second mTBI occurring

during this particular period of time (corresponding to the

period of brain vulnerability) may have catastrophic conse-

quences [9, 18, 34, 61, 62]. In this light, it is worth recalling

that previous studies indicated a varied response across cell

types and cellular viability based on repeat insult [34] or

post-stretch injury exposure to the inflammatory milieu [63],

thus confirming that cells experiencing an mTBI enters into a

temporary state of metabolic vulnerability.

A further implication of the present results concerns the

development of pharmacological interventions for mTBI.

For instance, according to the data showing downregula-

tion of genes controlling the ETC and the ATP biosyn-

thesis, potential therapies aimed at optimizing cell

metabolism should allow mildly injured cells to achieve a

net energy gain with no increase in mitochondrial function.

Data presented in this report represent a snapshot of an

evolving and rapidly adapting situation; therefore, further

studies aimed at characterizing the detailed time course of

modified gene expression following mild injury are nec-

essary, in particular with regard to the window of vulner-

ability and the possible therapeutic interventions.

Acknowledgments We wish to thank the Wessex Medical Research

Centre for funding this study and for their support to our research. We

also wish to thank Ms. Kathryn Rasco for her technical assistance in

the manuscript preparation.

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Potentially neuroprotective gene modulation in an in vitro model of mild traumatic brain injury

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