Neuropeptides

Neuropeptides 38 (2004) 40–47

www.elsevier.com/locate/npep

Neurogenic inflammation is associated with developmentof edema and functional deficits following

traumatic brain injury in rats q

A.J. Nimmo a, I. Cernak b, D.L. Heath a, X. Hu a, C.J. Bennett a, R. Vink b,c,*

a School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Qld., Australiab Department of Neuroscience, Georgetown University, Washington, DC, USA

c Department of Pathology, University of Adelaide, Adelaide, SA 5005, Australia

Received 27 October 2003; accepted 20 December 2003

Abstract

The present study has used capsaicin-induced neuropeptide depletion to examine the role of neurogenic inflammation in the

development of edema and functional deficits following traumatic brain injury (TBI). Adult, male rats were treated with capsaicin

(neuropeptide-depleted) or equal volume vehicle (controls) 14 days prior to induction of moderate/severe diffuse TBI. Injury in

vehicle treated control animals resulted in acute (4–5 h) edema formation, which was confirmed as being vasogenic in origin by

diffusion weighted magnetic resonance imaging and the presence of increased permeability of the blood–brain barrier (BBB) to

Evans blue dye. There was also a significant decline in brain magnesium concentration, as assessed by phosphorus magnetic res-

onance spectroscopy, and the development of profound motor and cognitive deficits. In contrast, capsaicin pre-treatment resulted in

a significant reduction in post-traumatic edema formation (p < 0:001), BBB permeability (p < 0:001), free magnesium decline

(p < 0:01) and both motor and cognitive deficits (p < 0:001). We conclude that neurogenic inflammation may play an integral role in

the development of edema and functional deficits following TBI, and that neuropeptides may be a novel target for development of

interventional pharmacological strategies.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Neurotrauma; Capsaicin; Diffuse brain injury; Edema; Magnesium; Neuropeptides

1. Introduction

Traumatic brain injury (TBI) remains the major killer

of individuals under 45 years of age. In younger victims

of brain trauma, edema and brain swelling is responsible

for 50% of all deaths (Feickert et al., 1999). Survivors

are often left with debilitating functional deficits thatsignificantly impacts on their quality of life. Although

the mechanisms associated with post-traumatic edema

formation are unclear, studies of peripheral tissue injury

have demonstrated that neuropeptides are associated

qSupported, in part, by the Australian National Health and Medical

Research Council and the Australian Research Council.* Corresponding author. Tel.: +61-8-8222-3092; fax: +61-8-8222-

3093.

E-mail address: robert.vink@adelaide.edu.au (R. Vink).

0143-4179/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.npep.2003.12.003

with development of increased vascular permeability

and edema formation through a process known as

neurogenic inflammation (Woie et al., 1993). Neuro-

genic inflammation is a neurally elicited reaction that

has typical characteristics of an inflammatory reaction

including vasodilatation, protein extravasation and tis-

sue swelling. Studies of peripheral nerves have demon-strated that neurogenic inflammation is the result of the

stimulation of C-fibers that causes the release of neu-

ropeptides (Woie et al., 1993). These neuropeptides

cause plasma protein extravasation from blood vessels

and associated edema formation. Although a number of

neuropeptides have been implicated in this process, it is

generally accepted that substance P (SP) increases mi-

crovascular permeability leading to edema formationwhilst calcitonin gene related peptide (CGRP) is an ex-

tremely potent vasodilator (Newbold and Brain, 1995).

A.J. Nimmo et al. / Neuropeptides 38 (2004) 40–47 41

Virtually all blood vessels of the body are surrounded bysensory nerve fibers that contain both CGRP and SP.

Cerebral arteries, in particular, appear to receive a dense

supply of these neurons, and it is therefore consistent

that these neurons may have a role as mediators of the

inflammatory process. However, no study to date has

ever examined the possibility that neurogenic inflam-

mation may play a role in TBI, despite a clear recogni-

tion that inflammation is a major factor in thedevelopment of neuronal cell death (Arvin et al., 1996;

Morganti-Kossmann et al., 2002).

Although selective antagonists to the individual

neuropeptide receptors are available (Leroy et al., 2000;

Parsadaniantz et al., 2000), it is necessary to first es-

tablish that sensory neuropeptides in general play a role

in TBI. While simultaneous inhibition of all the sensory

neuropeptide receptors with these antagonists is not yetpractical or feasible, a number of studies have now

shown that the neuropeptides can be depleted from

sensory nerves using capsaicin. Activation of neuro-

peptide containing sensory nerves by capsaicin will in-

duce the release of both CGRP and SP (Dray, 1992;

Tjen-A-Looi et al., 1998). While single exposure to

capsaicin induces an acute neurogenic inflammatory

response, repeated administration of capsaicin to adultanimals results in a transient depletion of SP and CGRP

stores and the inhibition of neurogenic inflammation

lasting up to 3 weeks (Cadieux et al., 1986; Kashiba

et al., 1997). Induction of traumatic brain injury during

this phase of neuropeptide depletion would provide an

opportunity to establish whether neurogenic inflamma-

tion plays a potential role in the development of edema

and functional deficits following TBI. The current studyhas therefore examined the effects of capsaicin-induced

depletion of neuropeptides on blood–brain barrier

(BBB) permeability, edema formation and both motor

and cognitive outcome in rats following severe, diffuse

TBI.

2. Materials and methods

All experimental protocols were approved and con-

ducted according to the guidelines established for the

use of animals in experimental research as outlined by

the Australian National Health and Medical Research

Council.

2.1. Capsaicin pre-treatment and induction of injury

Adult male Sprague–Dawley rats (n ¼ 44; 400� 25 g)

were administered 125 mg/kg capsaicin in 10% alcohol,

10% Tween 80 and 80% saline by subcutaneous injection

over three days (25, 50 and 50 mg/kg). Control animals

were administered vehicle alone over the same three-day

period. This procedure has been previously shown to

induce a depletion of neuropeptides lasting for up to3 weeks (Kashiba et al., 1997). Neuropeptide depletion

after pre-treatment was confirmed using the eye-wipe

response to a dilute capsaicin solution (0.1% in saline) as

used clinically. At 14 days, capsaicin pre-treated animals

typically did not exhibit an eye-wipe response with vi-

sual exposure to the capsaicin solution whereas vehicle

treated animals exhibited a clear eye wipe response

within 5 s. Accordingly, animals were subjected to TBIat 14 days after capsaicin treatment. Briefly, animals

were anesthetized with isoflurane and injured by impact-

acceleration induced diffuse brain injury, whereby a

stainless steel disc (10� 3 mm) fixed centrally on the

exposed skull between lambda and bregma is struck by

an accelerating metal impactor, as previously described

by our laboratory (Heath and Vink, 1995; Cernak et al.,

2001). This form of injury has been shown to result indiffuse brain trauma with axonal injury and moderate to

severe neurological deficits (Foda and Marmarou, 1994;

Povlishock et al., 1997; Cernak et al., 2001). Rectal

temperature was maintained at 37 �C throughout using

a thermostatically controlled heating pad and blood

pressure was monitored in a subgroup of animals to

ensure similar responses amongst treatment groups.

A further 22 animals were subject to all surgical proce-dures used to induce trauma, but were not injured

(shams).

After injury, animals were assessed for either edema

formation by diffusion weighted magnetic resonance

imaging (DWI; n ¼ 6/group) or wet weight/dry weight

determinations (n ¼ 5/group), brain free magnesium

concentration by phosphorus magnetic resonance spec-

troscopy (MRS; n¼ 6/group), BBB integrity using Ev-ans blue dye penetration (n ¼ 5/group), and functional

motor and cognitive deficits by rotarod (n ¼ 6/group),

and Barnes maze (n ¼ 6/group).

2.2. Diffusion-weighted magnetic resonance imaging

At 4 h after injury, a subgroup of animals (n ¼ 6/

group) was reanesthetized with isoflurane and assessedfor edema formation by DWI as previously described

(Hanstock et al., 1994). A further 6 untreated, uninjured

animals were used as sham controls. Briefly, animals

were placed in a specially constructed, temperature

controlled plexiglass holder and positioned in the center

of the 7 T magnet bore where a 72 mm proton tuned

birdcage coil had been positioned. Field homogeneity

across the brain was then optimised, and a sagittal pilotscan obtained for accurate placement of eight coronal

slices from the olfactory bulb to the cerebellum, using a

1 mm inter-slice distance. DWI images were then ac-

quired with a spin echo pulse sequence that had diffu-

sion gradients added before and after the refocusing

pulse. Gradient strength was varied in six steps using

sensitization values ranging from 20 to 1000 s/mm2.

42 A.J. Nimmo et al. / Neuropeptides 38 (2004) 40–47

A 256� 256 matrix was used with a 4 cm field of view,TR 2.0 s, TE 502 ms, slice thickness of 2 mm and 4

echoes. Rectal temperature and respiration was moni-

tored at all times. Diffusion maps were generated by

applying the Stejskal–Tanner equation in association

with a Marquart algorithm using the commercially

available Paravision software (Bruker, Billerica, MA,

USA). Apparent diffusion coefficients (ADCs) were

calculated for four regions: left cortex, right cortex, leftsubcortex and right subcortex. ADC were expressed as

10�5 mm2/s� SEM.

2.3. Phosphorus magnetic resonance spectroscopy

Immediately prior to or following DWI (4–5 h post-

injury), animals were subject to phosphorus MRS as

previously described in detail elsewhere (Heath andVink, 1999). Briefly, animals were placed in the specially

constructed, temperature controlled plexiglass holder

and a 5 mm� 9 mm surface coil was placed centrally

over the exposed skull. Skin and muscle were retracted

well clear of the coil to prevent contributions from these

tissues. The animals were then inserted into the center of

a 7.0 T magnet interfaced with a Bruker spectrometer

and field homogeneity optimized on the water signalprior to acquisition of phosphorus spectra. Phosphorus

spectra were obtained in 20 min blocks using a 90� pulsecalibrated for a 2 mm cortical depth, a 800 ms delay

time, and a 6000 Hz spectral width containing 2048 data

points. Rectal temperature and respiration was moni-

tored at all times.

2.4. Assessment of brain water content and blood–brain

barrier permeability

For assessment of BBB integrity, Evans blue dye

(0.3 ml of 2% solution in phosphate buffered saline) was

injected into the penile vein at 4.5 h after trauma and the

animals (n ¼ 5/group) decapitated at 5 h post-injury.

The BBB permeability to Evans blue dye relative to

control traumatized animals was assessed by determin-ing light absorbance in transverse sections of the brains

using the Image Pro Plus image analysis computer

program. For quantitation of edema formation, animals

(n ¼ 5/group) were killed at 5 h by decapitation under

isoflurane anesthesia and their brains rapidly removed

and weighed. Brains were then dried for 48 h at 100 �Cand reweighed. Water content was then calculated and

expressed as a percentage of total brain weight.

2.5. Assessment of functional outcome

Functional outcome (n ¼ 6/group) was assessed using

the rotarod and Barnes Maze. Briefly, the rotarod test as

used in previous TBI studies (Hamm et al., 1994;

O�Connor et al., 2003) requires an animal to walk on a

motorized rotating assembly of 18 rods, each 1 mm indiameter. The rotational speed of the assembly is in-

creased from 0 to 30 revolutions per minute (rpm) in

intervals of 3 rpm every 10 s. The duration in seconds at

the point at which the animal either completed the 2 min

task, fell from the rods, or gripped the rods and spun for

two consecutive revolutions rather than actively walk-

ing, was recorded as the task score. Acute spatial

learning and memory was assessed using the Barnesmaze (Barnes, 1979; Fox et al., 1998) as previously de-

scribed by our laboratory (O�Connor et al., 2003).

Briefly, animals were placed under a cover in the center

of an elevated 1.2-m diameter board containing 19 holes

around the periphery. One of the holes is the entrance to

a darkened escape tunnel that was not visible from the

surface of the board. After activating a series of bright

lights and an aversive sound, the cover was lifted and thelatency in seconds for the animal to locate and enter the

darkened escape tunnel was recorded. For both func-

tional outcome tests, animals were pre-trained on the

tasks for 5 days prior to injury.

2.6. Data analysis

Phosphorus MRS spectra were analyzed using theresident Bruker computer software program to assign

chemical shifts. Brain intracellular free magnesium

concentration was then determined from the chemical

shift difference between the a and b peaks of ATP using

the equation

Mg2þ� �

¼ Kd

10:82� da�b

da�b � 8:35

� �; ð1Þ

where da�b is the chemical shift difference between the aand b peaks of ATP. The Kd for MgATP was initially

assumed to be 50 lM at pH 7.2 and 0.15 M ionic

strength and was corrected for pH according to Bock

and colleagues (1987). The brain pH was determined

from the chemical shift of the inorganic phosphate peakrelative to phosphocreatine as previously described

(Heath and Vink, 1999).

All data are expressed as means and SEM. Statistical

differences were determined using a one-way analysis of

variance followed by individual Student–Newman–

Keuls post hoc tests. A p value of 0.05 was considered

significant.

3. Results

ADC maps derived from the DWI are shown inFig. 1. The ADC map in the injured, vehicle treated

animals (Fig. 1(b)) clearly shows areas of hyperintensity

throughout the brain compared to that in the sham

(untreated and uninjured) control animals (Fig. 1(a)).

These areas of hyperintensity indicate less restricted

Fig. 1. Apparent diffusion coefficient maps derived from the diffusion

weighted magnetic resonance images obtained at 4–5 h after diffuse

traumatic brain injury in rats: (a) sham (na€ıve); (b) vehicle treated;

(c) capsaicin pre-treated. Areas of cortical and subcortical hyperin-

tensity in the vehicle treated animals indicates vasogenic edema.

A.J. Nimmo et al. / Neuropeptides 38 (2004) 40–47 43

diffusion of water compared to the controls. At this

early 4–5 h time point, it is unlikely that there is a sig-

nificant loss of diffusion barriers to water, and we con-

clude that these regions of hyperintensity reflect

vasogenic edema. This result is consistent with ourprevious DWI observations following TBI (Hanstock

et al., 1994). In contrast, animals that were neuropep-

tide depleted with capsaicin pre-treatment did not

demonstrate any significant edema development after

injury when compared to the vehicle treated animals

(Fig. 1(c)). Quantitation of these ADC differences is

shown in Fig. 2. Sham controls had a mean ADC value

in both cortical and subcortical regions of 72.68�1.43� 10�5 mm2/s, which is similar to previously pub-

LC LSC RC RSC0

20

40

60

80

100

120

140Sham Vehicle Capsaicin

Brain Region

*** *** *** ***

AD

C (

10-5

cm

2 /sec

)

Fig. 2. Apparent diffusion coefficients obtained at 4–5 h after diffuse

traumatic brain injury in rats (n ¼ 6/group; mean�SEM). LC – left

cortex; LSC – left subcortex; RC – right cortex; RSC – right subcortex.

(*** p < 0:001 versus vehicle treated animals).

lished results (Hanstock et al., 1994; Albensi et al.,2000). After injury, there was a highly significant (p <0:001) increase in ADC in both the cortex and

sub-cortex in vehicle treated animals (mean 112.13–

125.33� 10�5 mm2/s) indicating an increased diffusion

distance of water consistent with vasogenic edema. This

increase was significantly inhibited (p < 0:001) in the

capsaicin pre-treated animals, with ADC values never

exceeding 77.80� 2.19� 10�5 mm2/s (Fig. 2).The changes in edema observed by DWI were con-

firmed using wet weight/dry weight determinations of

brain water content. The percentage water content in

shams was 77.8� 0.2%, which is consistent with values

reported in rats by others (Kita and Marmarou, 1994;

Bareyre et al., 1997). At 5 h after injury, vehicle treated

control animals had a brain water content of

80.0� 0.4% (p < 0:001) indicating the development ofedema after severe, diffuse TBI. Treatment with capsa-

icin resulted in a profound reduction in edema at 5 h

post-trauma compared to vehicle-treated animals

(p < 0:001; Fig. 3(a)). The reduction in edema formation

was associated with a significant reduction in the per-

meability of the BBB at 5 h after injury as assessed by

penetration of Evans blue dye (Fig. 3(b)). Consistent

with the inability of Evans blue to cross the intact BBB,negligible penetration of the dye was observed in sham

(uninjured) animals. Trauma, on the other hand, in-

creased BBB permeability such that Evans blue entered

the brain parenchyma in vehicle treated animals. Cap-

saicin treatment resulted in a profound, 85% reduction

in Evans blue penetration compared to the vehicle

treated controls (p < 0:001). Since increased BBB per-

meability associated with edema at early time pointsafter TBI are thought to represent vasogenic edema

(Beaumont et al., 2000), these results confirm our DWI

results suggesting vasogenic edema formation between

4 and 5 h after diffuse TBI. Moreover, the reduction in

BBB permeability and vasogenic edema formation with

capsaicin pre-treatment confirms that release of neuro-

peptides, in the form of neurogenic inflammation, may

play a significant role in these events.Previous studies have shown that TBI results in a

significant decline in brain free magnesium concentra-

tion within the first few hours after injury (Heath and

Vink, 1999; Vink and Cernak, 2000). To ascertain

whether neuropeptide depletion affects magnesium de-

cline after injury, animals were subjected to phosphorus

MRS at 4–5 h after injury. Injury in vehicle treated

animals resulted in a significant (p < 0:05) decline inbrain free magnesium concentration as compared to

sham animals (Fig. 4). The free magnesium values and

post-traumatic decline are similar to our previously

published results in this model of injury (Heath and

Vink, 1995; Cernak et al., 2001). In contrast, capsaicin

pre-treated animals did not show a significant decrease

in brain free magnesium concentration by 4–5 h after

Sham Vehicle Capsaicin0.2

0.3

0.4

0.5

0.6

*

Intr

acel

lula

r F

ree

Mg

(m

M)

Fig. 4. Brain free magnesium concentration at 4–5 h after diffuse

traumatic brain injury in rats (n ¼ 6/group; mean� SEM; * p < 0:05).

Fig. 3. Changes in (a) brain water content and (b) BBB permeability to Evans blue dye (n ¼ 5/group) following diffuse traumatic brain injury in rats

(n ¼ 5/group; mean� SEM). There was a highly significant difference between capsaicin treated animals and vehicle treated control animals after

injury (*** p < 0:001).

44 A.J. Nimmo et al. / Neuropeptides 38 (2004) 40–47

injury. Indeed, their free magnesium concentration after

injury was 0.47� 0.03 mM, which is similar to the valuesobserved in sham (uninjured) control animals (Fig. 4).

0

20

40

60

80

100

120

Ro

taro

d S

core

(se

cs)

0 1 2 3 4 5 6

Time (days) Postinjury

***

***

***

**

(a)

Fig. 5. Changes in (a) rotarod motor score and (b) Barnes maze cognitive s

SEM). Animals were pre-trained for 5 days immediately prior to injury and t

capsaicin. There was a highly significant difference between capsaicin treated

** p < 0:01).

Similar protective effects of capsaicin pre-treatment

were observed in the functional experiments. Prior to

injury, mean rotarod score in all animals was 106� 4 s.

There was no difference between vehicle treated controls

and capsaicin treated animals during the pre-injury

training period suggesting that capsaicin treatment did

not affect motor performance. After injury, vehicletreated control animals demonstrated a highly signifi-

cant (p < 0:001) decline in rotarod score to a minimum

of 23� 6 s, and a gradual recovery over time back to

pre-injury values (Fig. 5(a)). This decline and recovery

of motor function after TBI is consistent with previous

results in this model of injury (Heath and Vink, 1999).

In contrast to the vehicle controls, capsaicin-treated

animals demonstrated a profound attenuation of post-traumatic motor deficits after TBI. Indeed, the rotarod

score in the capsaicin treated animals was never less

0

50

100

150

200

250

Bar

nes

Maz

e L

aten

cy (

secs

)

0 1 2 3 4 5

Time (days) Postinjury

***

***

***

***

6

(b)

core after diffuse traumatic brain injury in rats (n ¼ 6/group; mean�hen assessed daily after injury for 6 days. s – shams; � – vehicle; j –

animals and vehicle treated control animals after injury (*** p < 0:001;

A.J. Nimmo et al. / Neuropeptides 38 (2004) 40–47 45

than 98� 2 s, which is very similar to the sham (unin-jured) group. Similar results were observed in the cog-

nitive tasks (Fig. 5(b)). Prior to injury, all animals

typically took less than 20 s to escape the aversive

stimuli in the Barnes maze. After injury, vehicle treated

animals took significantly longer (p < 0:001) to locate

the escape tunnel, and then improved the escape latency

with repeated exposure to the task (Fig. 5(b)). In con-

trast, capsaicin treated animals never demonstrated anyincrease in escape latency after injury. Their perfor-

mance after injury was always equivalent to the sham

(uninjured) animals.

4. Discussion

In the present study, we demonstrate that depletionof neuropeptides prior to induction of TBI results in the

attenuation of post-traumatic BBB permeability, edema

formation and functional neurologic deficits. While a

role for neuropeptides in general has been recognized in

central nervous system injury for a number of years

(Faden, 1986), a role for sensory neuropeptides has not

been previously reported. There have, nonetheless, been

a number of reports that implicate these neuropeptidesas factors that may contribute to the injury process

(Mantyh et al., 1989; Sharma et al., 1990; Lin, 1995;

Malcangio et al., 2000; Stumm et al., 2001). Earlier re-

ports have shown that sensory neuropeptide binding

sites are expressed by glia following brain injury

(Mantyh et al., 1989), a response that has since been

associated with post-traumatic reactive gliosis (Lin,

1995). Significant amounts of sensory neuropeptide re-lease have subsequently been detected in the nervous

system following both peripheral nerve injury (Mal-

cangio et al., 2000) and traumatic spinal cord injury. In

brain ischaemia, activation of neuropeptide receptors in

the endothelium has been shown to contribute to edema

formation (Stumm et al., 2001), an event that has been

well characterised in peripheral tissue injury (Woie et al.,

1993). This observation is consistent with our presentresults, where induction of TBI resulted in the devel-

opment of vasogenic edema within the first few hours

after of injury. Moreover, prior depletion of sensory

neuropeptides with capsaicin resulted in an almost

complete inhibition of increased BBB permeability and

vasogenic edema formation after TBI, supporting a role

for neuropeptides in vasogenic edema formation fol-

lowing diffuse TBI.The magnetic resonance imaging (DWI) results in the

present study were also consistent with previous pub-

lished studies. The vasogenic edema formation in the

acute (4–5 h) period following TBI has been reported in

a number of experimental models including lateral fluid

percussion injury (Hanstock et al., 1994; Albensi et al.,

2000), closed cortical impact injury (Beaumont et al.,

2000) and in diffuse impact acceleration induced injury(Beaumont et al., 2000). This vasogenic edema is fol-

lowed by a gradual development of cytotoxic edema,

which can persist for a number of days after injury.

Notably, the early vasogenic edema is thought to be

permissive to the development of the cytotoxic compo-

nent of edema (Beaumont et al., 2000), suggesting that

inhibition of the early vasogenic phase may also atten-

uate the development of the later cytotoxic edema. Inthe present study, we have shown that neuropeptide

depletion inhibits the acute vasogenic edema, and our

own preliminary studies have shown that such treatment

also inhibits edema development at 24 h (Vink et al.,

2002). Further studies are required to establish whether

the inhibition of neurogenic inflammation also attenu-

ates edema formation after 24 h when the cytotoxic

phase reaches its nadir.Our results also demonstrate that neuropeptide

release may influence functional outcome after TBI.

Indeed, prior neuropeptide depletion with capsaicin pre-

treatment resulted in a profound attenuation of both

motor and cognitive deficits immediately after TBI.

There are high numbers of sensory neuropeptide re-

ceptors in the hippocampus and striatum, those parts of

the brain that are known to be associated with learningand memory, supporting a potential role for these neu-

ropeptides in learning and memory (Huston and Has-

enohrl, 1995). Subsequent studies have confirmed that

alterations in calcitonin-gene related peptide, substance

P and neuropeptide Y in the hippocampus are associ-

ated with cognitive deficits in mice (Bracci-Laudiero

et al., 1999). A role for sensory neuropeptides in neuro-

toxicity and motor function has also been well estab-lished (Gaumann et al., 1990) while recent clinical trials

have supported a role for neuropeptides, and particu-

larly substance P, in depression (Rupniak and Kramer,

1999). The present study has also shown that capsaicin

induced depletion of neuropeptides prior to injury re-

sulted in a profound attenuation of the magnesium de-

cline in the first 4–5 h. Decline in brain free magnesium

concentration after TBI has been extensively demon-strated using MRS in a number of experimental injury

models, including the impact acceleration model used in

the current study (for review, see (Vink and Cernak,

2000)). Moreover, this decline has been associated with

the development of functional deficits following trauma.

Taken together, a role for the sensory neuropeptides in

determining functional outcome following TBI therefore

seems feasible. Presumably, if this is the case, increasingneuropeptide release with injury may exacerbate injury.

Notably, we observed that induction of injury at 7 days

after capsaicin treatment in animals (while capsaicin

induced neurogenic inflammation was still ongoing) led

to a 100% mortality (unpublished results), most likely by

exacerbating any neurogenic inflammation that is asso-

ciated with TBI.

46 A.J. Nimmo et al. / Neuropeptides 38 (2004) 40–47

While previous studies using capsaicin have shownthat this form of treatment in adult animals depletes

sensory neuropeptides (Cadieux et al., 1986; Kashiba

et al., 1997; Tjen-A-Looi et al., 1998), there is the pos-

sibility that the reduced sensory neuropeptide activity

could be associated with receptor desensitization. This

is, however, unlikely given that our preliminary results

examining TBI in capsaicin treated neonatal animals

demonstrated the same protective effect as that seen inthe current adult study (Vink et al., 2002). Capsaicin

treatment in neonatal animals is well known to produce

permanent sensory neuropeptide depletion without re-

ceptor desensitization (Cuello et al., 1981; Levasseur

et al., 1993; Geraghty and Mazzone, 2003). Similarly,

the present study does not identify which neuropeptides

are associated with the development of edema and

functional deficits after trauma. Nonetheless, it has beenpreviously shown that both substance P and CGRP are

depleted by capsaicin treatment (Dray, 1992; Kashiba

et al., 1997). Moreover, it is widely accepted that SP

increases microvascular permeability leading to edema

formation whilst CGRP is an extremely potent vasodi-

lator (Newbold and Brain, 1995). Since we observed a

decrease in vascular permeability and edema formation

with injury induced after capsaicin pre-treatment, wewould speculate that substance P is worthy of more at-

tention as an injury factor following traumatic brain

injury. Future studies will examine the potential of se-

lective substance P antagonists as a therapeutic inter-

vention after traumatic brain injury.

In conclusion, we have demonstrated that inhibition

of neurogenic inflammation by prior depletion of neu-

ropeptides with capsaicin treatment inhibits BBBopening, edema formation, free magnesium decline and

development of motor and cognitive deficits after TBI.

These results suggest that sensory neuropeptides may

play a significant role in the post-traumatic, secondary

injury process and may offer a novel target for devel-

opment of interventional pharmacological strategies.

Acknowledgements

We thank Jill MacLeod for excellent technical

assistance.

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