Technical Aspects of an Impact Acceleration Traumatic Brain Injury Rat Model with Potential Suitability for both Microdialysis and PtiO2 Monitoring

Technical Aspects of an Impact Acceleration Traumatic Brain Injury
Rat Model with Potential Suitability for both Microdialysis and PtiO2
Monitoring.

Emilie Carré 1 *, Emmanuel Cantais 2, Olivier Darbin 3, Jean-Pierre Terrier 4,
Michel Lonjon 5, Bruno Palmier 2, Jean-Jacques Risso 1.

1 Department of Neurochemistry, I.M.N.S.S.A., B.P. 610, 83800 Toulon Armées,
France.
2 Intensive Care Unit, H.I.A. Sainte-Anne, B.P. 600, 83800 Toulon Armées, France.
3 Department of Neurology, School of Medicine, Springfield, IL, USA.
4 Department of Pathology, H.I.A. Sainte-Anne, B.P. 600, 83800 Toulon Armées,
France.
5 Department of Neurosurgery, CHU Pasteur, Nice, France.

20 text pages and 3 figures

* Corresponding author:
Emilie Carré, Department of Neurochemistry, I.M.N.S.S.A., B.P. 610, 83800 Toulon
Armées, France.
Tel.: (+33) 494099630
Fax: (+33) 494099251
E-mail: emilie_carre@hotmail.com

1

---

Author's full names

Author to whom proofs and correspondence are to be sent:
Emilie Carré
Department of Neurochemistry, I.M.N.S.S.A., B.P. 610, 83800 Toulon Armées, France.
Tel.: (+33) 494099630
Fax: (+33) 494099251
E-mail: emilie_carre@hotmail.com

(or j.j.risso@imnssa.net for heavy files)


Emmanuel Cantais and Bruno Palmier, Intensive Care Unit, H.I.A. Sainte-Anne,
83800 Toulon Armées, France.

Olivier Darbin, Department of Neurology, Southern Illinois University School of
Medicine, P.O. Box 19637, Springfield, IL 62794, USA.

Jean-Pierre Terrier, Department of Pathology, H.I.A. Sainte-Anne, B.P. 600, 83800
Toulon Armées, France.

Michel Lonjon, Department of Neurosurgery, CHU Pasteur, 30 avenue de la voie
romaine, B.P. 69, 06002 Nice cedex1, France.

Jean-Jacques Risso, Department of Neurochemistry, I.M.N.S.S.A., B.P. 610, 83800
Toulon Armées, France.

2

---

Abstract
This report describes technical adaptations of a traumatic brain injury model – largely
inspired by Marmarou – in order to monitor microdialysis data and PtiO2 (brain tissue
oxygen) before, during and after injury. We particularly focalize on our model
requirements which allows us to re-create some drastic pathological characteristics
experienced by severely head-injured patients: impact on a closed skull, no ventilation
immediately after impact, presence of diffuse axonal injuries and secondary brain
insults from systemic origin… We notably give priority to minimize anaesthesia
duration in order to tend to banish any neuroprotection.
Our new model will henceforth allow a better understanding of neurochemical and
biochemical alterations resulting from traumatic brain injury, using microdialysis and
PtiO2 techniques already monitored in our Intensive Care Unit. Studies on efficiency and
therapeutic window of neuroprotective pharmacological molecules are now conceivable
to ameliorate severe head-injury treatment.

Keywords: Traumatic Brain Injury, Microdialysis, PtiO2, Weight-drop, Impact
Acceleration, Diffuse Axonal Injury, Neuroprotection, Methodology.



3

---

1. Introduction
In order to explain the microdialysis and PtiO2 (brain tissue oxygen) data monitored on
severely head-injured patients from our Intensive Care Unit and to study the specific
effects of anaesthesia and/or neuroprotection, a traumatic brain injury (TBI) animal
model, similar to human head injury, is indispensable.
According to Bullock et al. (1999), "individual animal models rarely, if ever, model the
entire spectrum of pathological characteristics observed in the patient population with
severe head injuries". So the majority of drugs that have shown a neuroprotective effect
on animal have usually few effects on severely head-injured patients. For this reason we
have chosen to give priority to the concordance between our traumatized animals and
head-injured patients characteristics.
The Marmarou impact acceleration model, commonly called “weight-drop”, is a well-
known model of traumatic brain injury. This model, precisely described in two
complementary articles (Marmarou et al., 1994 ; Foda and Marmarou, 1994), compiles
some of the most important characteristics of human head-injury. Because of its closed
skull impact, this model is more particularly in agreement with the cases of falls or road
accidents, which are the most frequent situations in our Intensive Care Unit. Moreover
this model was fully attested to produce diffuse axonal injury similar to that described
in man – these diffuse axonal injuries are detected in more than 90% of fatal head
injured patients (Gentleman et al., 1995).
As far as we know, microdialysis and PtiO2 monitoring have never been envisaged
before and during impact in this type of model. So we report, in this article, some
technical aspects of the TBI model adaptations to this intracerebral concomitant
monitoring before, during and after impact. We particularly focalize on the

4

---

requirements of our model – especially the limitation of neuroprotection - in order to re-
create the most drastic conditions inherent in human head injury.


5

---

2. Materials and Methods
Ethics
The experimental protocol was approved by the local ethics committee and by the
French Ministry of Defence.
2.1. Specificities of the impact acceleration TBI model
Preparation of intracerebral guides and PtiO2 probe
Before any surgery, 2 intracerebral guides (CMA, Phymep, Paris), the first devoted to
microdialysis and the second devoted to PtiO2, are bonded together with a specific
angle, using Araldite glue (Bostik S.A., France). This angle should be determined so
that sensitive areas of both microdialysis and PtiO2 probes membranes will be very close
but not directly in contact.
Because no introducer seems to be available for PtiO2, a modified dummy cannula is
glued on the PtiO2 probe so that the probe will perfectly fit to the guide.
Surgery
Two weeks before the experimentation, a male Sprague-Dawley rat weighting between
400 and 450g (OFA strain, Iffa Credo, France) is anaesthetised with sodium
pentobarbital (60mg/kg intraperitoneally) before surgery.
The intracerebral microdialysis guide - bonded to the PtiO2 guide - is stereotaxically
implanted in the striatum, 4 mm higher than the precise location of the microdialysis
probe membrane, according to the atlas of Paxinos and Watson (1982): coordinates
relative to Lambda in mm A:+9.7; L:+3; H:+6.5. The two guides, and a support for
fixing to the swivel, are fixed on the very anterior part of the skull with two screws and
dental cement (Dentalon plus, Heraeus Kulzer, Germany). Immediately back, the
10mm-diameter impact site on the skull must absolutely stay clear of cement. The skin

6

---

above this impact site must be closed by stitches in order to be preserved from air
contact.
After surgery, the rat is allowed to recover in individual cage, under a 12-hours
light/dark cycle (light on from 07h00 to 19h00), with free access to food and water for
the two weeks before the experiment.
Microdialysis and PtiO2 monitoring
Twelve hours prior to the experiment, the rat is placed in the experimental plexiglass
cage in order to adapt to his new environment.
The day of the injury, the dummy cannula of the straight guide is replaced by the
microdialysis probe (CMA12, 4mm-length, 0.5mm-diameter, Phymep, France). This
probe is perfused by an artificial CSF (in mM ; NaCl:147 ; KCl:2.7 ; CaCl2:1.2 ;
MgCl2:0.85) at the rate of 1µL/min. The modified PtiO2 probe (LICOX CC1.R, 4mm-
sensitive area length, 0.5mm-diameter, Integra NeuroSciences, France) is inserted in the
slope guide and connected to the LICOX CMP instrument (Integra NeuroSciences,
France).
Because rat needs to be in freely-moving, a microdialysis liquid swivel has been
especially adapted in our lab in order to allow concomitant passing of both electrical
PtiO2 signal and liquid microdialysis samples.
A first phase of parameters stabilisation (at least 2 hours) should be observed. Then
basal levels of PtiO2 and microdialysis parameters can be monitored.
Induction of Traumatic Brain Injury
Preliminary experiments lead us to induce Traumatic Brain Injury under transient 3%-
isoflurane anaesthesia (less than 10 min).


7

---

Stitches above site impact are removed and a 10mm-diameter 3mm-thick metallic disk
– designed to protect against skull fracture - is placed directly in contact with the clear
part of the skull. A cylindrical metallic 430g-weight is dropped from two meters
through a metal tube onto the disk (Fig.1).

1
2
5
6
4
3
Fig.1: Schematic representation of the impact
acceleration device.
1
metal 2m-tube
2 430g-weight foam
3
bed
4
cement and impact site
5 velocity sensor
6
system preventing weight-rebound
(Figure freely adapted from Marmarou, 1994)


8

---

Weight rebound is prevented using an automated system especially designed in our lab.
A velocity sensor, designed in our lab as well, certifies reproducibility of the weight
acceleration at the very moment of the impact.
After impact, the rat is allowed to return to his plexiglass cage, in order to recover from
anaesthesia and to continue the recordings in conscious freely-moving conditions.

2.2. Evidences of traumatic brain injury
Behavioural test
Four days after injury, an adhesive-removal somatosensory test, modified from
Schallert et al. (2000), was used to reveal evidences of TBI-induced behavioural lesions.
A standardised adhesive stimulus was attached to one of the rat forelimb. Rats removed
the stimulus using their teeth. The latency of stimulus contact and removal was recorded
and compared between 6 injured and 6 sham-lesioned rats (non parametric C1 Fisher-
Yates-Terry test).
Brain Fixation and Histopathological Preparation
At least 24h after impact, rats were deeply anaesthetised with an intraperitoneal
injection of sodium pentobarbital. The chest was rapidly opened, a catheter was
introduced into the ascending aorta, and the right atrium was incised. Firstly, 200 mL of
heparin saline (1000U.I. of heparin in saline) were perfused through the catheter, at a
rate of 25mL/min. Secondly, 400 to 500mL of fixative (4% formaldehyde, 3% acid
acetic, in saline) were perfused at the same rate. The brain was carefully removed and
stored in a fixative (4% formaldehyde in saline) for at least 24 hours. The brain was
finally placed in 10%-formaldehyde for 12 hours before gross examination. Brain
coronal and sagittal sections were embedded in paraffin. Sections 5 µm-thick were cut

9

---

with a rotary microtome, stained with haematoxylin-eosin-safran (HES), and examined
under light microscopy.


10

---