Download Differential responses in three thalamic nuclei in moderately

DOI: 10.1093/brain/awh294
Brain (2004), 127, 2470–2478
Differential responses in three thalamic nuclei in
moderately disabled, severely disabled and
vegetative patients after blunt head injury
William L. Maxwell,1 Kyla Pennington,1 Mary Anne MacKinnon,2 Douglas H. Smith,4
Tracy K. McIntosh,4 J. T. Lindsay Wilson3 and David I. Graham2
1
Laboratory of Human Anatomy, Thomson Building,
Institute of Biomedical and Life Sciences, University of
Glasgow, 2Academic Unit of Neuropathology, Institute of
Neurological Sciences, Southern General Hospital,
Glasgow, 3Department of Psychology, University of Stirling,
Stirling, UK and 4Head-Injury Research Centre, Division of
Neurosurgery, University of Pennsylvania, Philadelphia,
USA
Correspondence to: William L. Maxwell DSc, Thomson
Building, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow G12 8QQ, UK
E-mail: [email protected]
Summary
In vivo imaging techniques have indicated for many years
that there is loss of white matter after human traumatic
brain injury (TBI) and that the loss is inversely related to
cognitive outcome. However, correlated, quantitative evidence for loss of neurons from either the cerebral cortex or
the diencephalon is largely lacking. There is some evidence
in models of TBI that neuronal loss occurs within the
thalamus, but no systematic studies of such loss have been
undertaken in the thalamus of humans after blunt head
injury. We have undertaken a stereological analysis of
changes in numbers of neurons within the dorsomedial,
ventral posterior and lateral posterior thalamic nuclei in
patients assessed by the Glasgow Outcome Scale as moderately disabled (n = 9), severely disabled (n = 12) and vegetative (n = 10) head-injured patients who survived between
6 h and 3 years, and controls (n = 9). In histological sections
at the level of the lateral geniculate body, the cross-sectional
area of each nucleus and the number and the mean size of
neurons within each nucleus was quantified. A statistically
significant loss of cross-sectional area and number of neurons occurred in the dorsomedial nucleus in moderately
disabled, and both the dorsomedial and ventral posterior
thalamic nuclei in severely disabled and vegetative headinjured patients. However, there was no change in neuronal
cell size. In the lateral posterior nucleus, despite a reduction
in mean cell size, there was not a significant change in either
nuclear area or number of neurons in cases of moderately
disabled, severely disabled or vegetative patients. We posit,
although detailed neuropsychological outcome for the
patients included within this study was not available, that
neuronal loss in the dorsomedial thalamus in moderately
and severely disabled and vegetative patients may be the
structural basis for the clinical assessment in the Glasgow
Outcome Scale. In severely disabled and vegetative patients, loss of neurons from the ventral posterior thalamic
nucleus may also reflect loss of response to afferent stimuli.
Keywords: human thalamus; stereology; traumatic brain injury; thalamus; loss of neurons
Abbreviations: DMN = dorsomedial nucleus; GOS = Glasgow Outcome Scale; LPN = lateral posterior nucleus;
TBI = traumatic brain injury; VPN = ventral posterior nucleus
Received May 18, 2004. Revised June 25, 2004. Accepted June 29, 2004. Advanced Access publication September 29, 2004
Introduction
After traumatic brain injury (TBI), there may be impairment
of cognitive and memory functions in both humans (Levine
et al., 2002) and experimental animals (Smith et al., 1997;
Pierce et al., 1998). In experimental animals, tissue loss from
Brain Vol. 127 No. 11
#
cerebral cortex, thalamus and the hippocampus has been correlated with the above behavioural changes. However, such
tissue loss has only been estimated in terms of changes in area
for cortical and hippocampal pyramidal cells (Smith et al.,
Guarantors of Brain 2004; all rights reserved
Thalamic responses after human blunt head injury
1997) not for total changes in area of specific brain regions.
In humans, although detailed analyses of neuropathological
changes after TBI are available (Graham et al., 2002), these
have focused on a description of the cellular responses and
their time course. Quantitative data for alteration in numbers
of neurons and glia within specific regions of the human brain
after TBI are largely lacking. However, recent studies have
indicated that damage within the thalamus is a correlate of
the degree of disability after TBI (Adams et al., 2000,
2001) and that damage within the thalamus may differ
between moderately disabled, severely disabled and vegetative patients (Jennett et al., 2001). Moreover, a difference in
clinical outcome has become established where patients who
are vegetative after blunt head injury are more likely to
experience some recovery than those who are vegetative
due to a non-traumatic insult, for example near fatal cardiac
arrest (Multi-Society Task Force, 1994).
In the thalamus, there is a consensus that the ventral posterior nucleus (VPN) is both the major site of termination for
nerve fibres forming the medial lemniscus/dorsal column
pathway, spinothalamic tract and the trigeminal cranial
nerve, and the origin of fibres to the primary somatic sensory
areas of the cerebral cortex; that neurons in the lateral posterior nucleus (LPN) project to the cortex of association sensory
areas posterior to the postcentral gyrus (Brodmann area 5);
and that the dorsomedial nucleus (DMN) is both the terminal
field of fibres from the olfactory cortex and amygdala, and the
origin of fibres to large areas of the frontal cortex and the
terminal field of reciprocal corticothalamic fibres (reviewed
by Jones et al., 1985). The complex functions of the DMN are
indicated by loss of motivational drive, loss of the ability to
solve problems and changes in the level of consciousness after
disease or injury.
Recent quantitative evidence for differential loss of hippocampal pyramidal neurons in subfields of the hippocampus
with increasing, post-traumatic survival after human blunt
head injury (Maxwell et al., 2003) has been provided. We
have extended that study to test the hypothesis that there is
loss of neurons from three thalamic nuclei with survival after
TBI. The present study used stereological techniques to
estimate numbers of neurons within the VPN, LPN and
DMN in moderately disabled, severely disabled and vegetative patients after TBI.
Methods
Preparation of samples
Post-mortem material was obtained from the paraffin-embedded
tissue archive held in the Department of Neuropathology, Southern
General Hospital from cases that had been assessed by the Glasgow
Outcome Scale (GOS) and had undergone a full neuropathological
examination (Adams et al., 2001; Jennett et al., 2001) (Table 1).
All material had been handled with care to minimize the incidence
of ‘dark’ neurons (Cammermeyer, 1961) by fixing all brains in 10%
formal saline for at least 3 weeks prior to dissection. Blocks were
obtained from thalamus in nine controls that had no history of head
2471
injury prior to death (mean age 35 years, range 18–60); nine
moderately disabled patients (mean age 36 years, range 19–60,
with survivals between 3 and 22 years after admission); 12 severely
disabled patients (mean age 40 years, range 23–70, with survivals
between 4 weeks and 8 years) and 10 vegetative patients (mean age
39 years, range 18–64, with survivals between 3 and 27 months).
All but two of the severely disabled and vegetative patients died
due to respiratory complications and/or pneumonia (Table 1). Variance between experimental groups differed both between groups
and between thalamic nuclei within groups. Variance was greatest
in the moderate disability group (see Table 2). This is not unexpected since these patients survived for years after the initial injury
and their post-traumatic life style may have influenced outcome in
terms of changes in number of neurons (Adams et al., 2001). The
present study utilized all available head injury material held within
the Glasgow Archive. Power analysis indicated a sample size of 38
patients for the DMN and VPN in moderate disability patients for
a value for P < 0.05, and 62 patients for the LPN. This number of
patients was not available. For severely disabled and vegetative
patients, a minimum of eight patients was required. That number of
patients was available within the Glasgow Archive. One member
of the research team coded the cases which were then examined
‘blind’ to clinical and pathological data by other members of the
research group. The Research Ethics Committee of the Southern
General Hospital approved this study.
Blocks 1 cm thick were obtained from the thalamus after routine
brain cut procedures in the coronal plane (Graham et al., 1995).
Serial sections (n = at least 10) of the left thalamus were cut at
7 mm and stained with cresyl violet/Luxol fast blue (Kluver
and Barrera, 1953) which allowed for the delineation of nuclear
boundaries within the thalamus.
After identification of the VPN, LPN and DMN in each section,
a graticule was placed randomly over the field of each nucleus and
the number of pyramidal neurons containing a discrete nucleolus
within each nucleus counted. When using the optical disector
method, between 100 and 150 cells need to be counted in order
to obtain an accurate estimate of the total number of objects (West
et al., 1991). However, stereological techniques assume that the
particles to be counted follow a Poisson distribution and that sections of 50–80 mm thickness are used. Neither of the above was true
for the tissue in the present study. First, the spatial distribution of
neurons in the brain varies systematically in different regions
(Williams and Rakic, 1988). Secondly, material was collected
over a 20–30 year period and all had been wax embedded for
routine neuropathology.
Three important factors influence a counting protocol. These are
(i) the size, orientation and shape of the object counted; (ii) the
numerical density of these objects; and (iii) the size of the sample
window and the 3D spatial distribution of the objects (Williams and
Rakic, 1988; Benes and Lange, 2001). To estimate the size of neurons within the selected nuclei, one section from each of the agematched controls was picked at random, using a random number
generator, from the 10 sections available. Five neurons in which
the nucleus contained a discrete nucleolus were selected from
each of the VPN, LPN and DMN, and two perpendicular transverse
diameters of each neuron measured using a calibrated micrometer.
There was a significant difference in the diameter of neurons within
each of the nuclei. The largest neurons (diameter of 14 6 2.21 mm)
were present in the VPN. This mean cell soma size allowed determination that the focus depth of the counting box, in order to minimize any possibility of the same cell being counted twice (Howard
2472
W. L. Maxwell et al.
Table 1 Clinical data for patients included in the study
Patient
Control
C1
C1
C3
C4
C5
Age
(years)
Sex
Cause
PMI
Survival
Skull
fracture
patient group (no history of head injury during life)
60
M
9D
NR
NR
18
M
8H
NR
NR
50
F
23H NR
NR
50
M
21H NR
NR
57
M
19H NR
NR
C6
38
M
C7
33
M
C8
62
M
C9
24
M
Moderate disability patient group
1
49
M
Fall
2
54
M
Fall
3
59
M
Fall
4
38
M
NK
5
37
M
Fall
6
19
M
ASS
7
60
M
NK
8
58
M
RTA
9
48
M
RTA
Severe disability patient group
1
23
M
ASS
2
76
M
Fall
3
70
F
Fall
4
51
M
RTA
5
60
M
RTA
6
74
F
RTA
7
55
M
RTA
8
65
M
NK
9
39
M
ASS
10
67
M
Fall
11
29
M
ASS
12
28
M
ASS
Vegetative patient
1
31
2
42
3
58
4
50
5
49
6
64
7
27
8
18
9
61
10
56
group
M
M
M
M
M
M
M
M
M
M
RTA
ASS
RTA
ASS
ASS
ASS
NK
ASS
NK
ASS
ICH
TCI
TAI
Diffuse
IBD
Raised
ICP
Cause of death
NR
NR
NR
NR
NR
–
–
–
–
–
–
–
–
–
–
NR
NR
NR
NR
NR
–
–
–
–
–
5D
56H
4D
7D
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
–
–
–
–
–
–
–
–
NR
NR
NR
NR
–
–
–
–
Myocardial infarction
Hodgkin’s disease
Carcinoma of breast
Broncho-pneumonia
Non-Hodgkin’s
lymphoma
Heart failure
Acute tracheobronchitis
Myocardial infarction
Drug overdose
23H
5D
23H
5D
6H
NK
5D
21H
17H
3Y
3Y
4Y
4Y
4Y
5Y
12Y
16Y
22Y
No
Yes
NK
NK
Yes
Yes
Yes
Yes
NK
No
ICHa
ICHa
No
ICHa
SDH
SDHa
EDH
SDHa
6
27
15
16
27
6
10
12
6
No
1
No
No
No
No
1
1
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
No
Yes
No
No
No
No
SUDEP
Myocardial infarction
GI haemorrhage
SUDEP
Pneumonia
SUDEP
Acute head injury
SUDEP
SUDEP
63H
46H
17H
17H
37H
63H
37H
4D
5H
54H
4D
5D
4W
5W
7W
2M
3M
3M
3M
4.5M
6M
8M
7Y
8Y
No
Yes
No
Yes
No
No
No
Yes
No
Yes
Yes
No
No
ICH
SDHa
SDHa
ICHa
SDHa
No
No
No
SDHa
SDHa
SDHa
0
18
8
6
3
12
16
0
0
13
3
0
2m
1
No
1
No
No
3 Mm
1
2m
No
No
No
+
No
+
No
No
+
+
No
No
++
+
++
No
No
No
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Empyema
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Acute respiratory
distress syndrome
52H
7H
17H
13H
37H
67H
26H
13H
4D
14H
6M
9M
9M
1Y
1Y
1Y 6M
1Y 6M
2Y
2Y 3M
3Y
No
No
No
No
No
Yes
No
No
No
No
No
No
No
SDHa
SDHa
No
No
No
B FP
No
6
0
0
4
9
3
0
6
6
0
3 Mm
3 Mm
3m
No
No
2M
3 Mm
3 Mm
No
3M
No
No
No
+
++
No
No
No
++
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Pneumonia
Pneumonia
Pneumonia
Pneumonia
Pyelonephritis
Pyelonephritis
Pneumonia
Pneumonia
Pneumonia
Pneumonia
M = male; F = female. Cause of injury: RTA = road traffic accident; ASS = assault; NK = not known; PMI = post-mortem interval [H = hours;
D = days. NR not relevant; B = bilateral, F = frontal; P = parietal. Survival: W = weeks; M = months, Y = years. EDH = extradural
haematoma, ICH = intracranial haematoma; SDHa = subdural haematoma evacuated; TCI = total contusion index (Adams et al., 1985);
TAI = diffuse (traumatic) axonal injury (Adams et al., 1989); IBD = diffuse ischaemic brain damage (Graham et al., 1989); ICP = raised
intracranial pressure (Adams and Graham, 1976).
and Reed, 1998; West and Gunderson, 1990), should be 28 mm. Cells
were counted in the first and fourth serial sections (separation of
counting planes 28 mm). The first slide in the whole series from each
patient was chosen using a random number generator for a number
between 1 and 5. Cells were counted in that section and the next
fourth section.
Estimation of the area of thalamic nuclei
On rostro-caudal sections of the thalamus, boundaries of the DMN,
VPN and LPN were visualized: the internal medullary lamina separating the DMN from the centromedian and lateral thalamic nuclei;
the LPN between the dorsal one-third of the internal medullary
lamina and the external medullary lamina; and the VPN between
Thalamic responses after human blunt head injury
2473
Table 2 Cross-sectional area (mm2) of each named thalamic nucleus, at the level of the lateral geniculate body,
and total number of neurons within a 28 mm thick slice in the different patient groups
Area in mm2
Control
DMN
48.26 6 3.2
LPN
35.49 6 3.2
VPN
45.36 6 2.3
Moderate disability head injury
DMN
47.59 6 6.3
PN
34.3 6 4.2
VPN
44.73 6 4.8
Severe disability head injury
DMN
35.41 6 2.8
LPN
33.8 6 4.7
VPN
40.67 6 2.4
Vegetative head injury
DMN
34.2 6 2.6
LPN
31.99 6 5.2
VPN
39.75 6 1.2
D% Change from control:
statistical significance
No. of neurons in a
28 mm thick slice
D% Change from control:
statistical significance
–
–
–
1822.99 6 100.0
1074.92 6 150.0
1528.02 6 77
–
–
–
1.38: P = 0.76
3.35: P = 0.48
1.38: P = 0.70
1732.83 6 123
998.93 6 198
1478 6 83
4.99: P = 0.04
7.07: P = 0.34
3.27: P = 0.18
26: P < 0.0001
4.76: P = 0.39
10.3: P = 0.0005
1453.09 6 178
968 6 203
1278 6 75
20.29: P < 0.0001
9.95: P = 0.19
16.36: P = 0.03
29.1: P < 0.0001
9.8: P = 0.08
12.4: P = 0.0001
1253.08 6 150
957.1 6 240
1259.3 6 69
31.2: P < 0.0001
10.88: P = 0.19
17.60: P = 0.015
The percentage change (D%) from control values and the P value for this change for each nucleus are also provided.
the external medullary lamina and the centromedian and ventral
posteromedial nuclei. A graticule, 1 mm 3 1 mm, was laid over
each nucleus and the section examined using a binocular dissecting
microscope, magnification 3 2. Standard stereological techniques
were used to count the total number of points formed by intersections
at the edges of squares of the graticule (point counting technique;
Howard and Reed, 1998; Mouton, 2002). At points at which the
boundary of a nucleus crossed the inclusion lines of a counting
frame (upper and right edges of the frame), points were counted,
whereas if the boundary crossed the exclusion lines (left and bottom
lines of the frame), points were not counted.
Estimation of the number of neurons
Within the series of slides obtained from each block, a randomly
(random number between 1 and 5 using a random number table)
selected section of the VPN, LPN and DMN in each patient was
identified. Recent work has indicated that it is technically more
efficient to use a larger counting window within the CNS (Benes
and Lange, 2001; Maxwell et al., 2003). In the present study,
a counting window with sides 250 3 250 mm was used. The number
of squares of the above dimensions that overlay each nucleus was
determined (773 for control DMN, 568 for control LPN and 742
for control VPN). A random number generator was used to obtain
a number between 1 and 20, and that numbered square, starting
from the top left hand corner of each thalamic nucleus, was used
for the first counting window. The total number of neurons within
which a discrete nucleolus was present within that window, using
standardized exclusion criteria (see previous paragraph), was
counted. The counting process was then repeated in every 20th
successive square across the entire cross-section of each of the
VPN, LPN and DMN. This provided a sample of 46 for the
DMN, 35 for the LPN and 47 for the VPN in controls, and 32–39
samples for each nucleus in the head-injured patients. To preclude
one nucleus being counted more than once, the number of nuclei
containing a discrete nucleolus was counted in the first and fourth
successive sections. Each counting box therefore contained a volume
of 0.00175 mm3. The mean number of neurons multiplied by the total
number of squares overlying each of the above nuclei gave the
number of neurons in a coronal slice of thickness 28 mm.
In this type of analysis, the coefficient of variance (CV), i.e. the
variation among a group, is the SD/mean. For accurate sampling
(West et al., 1991), the square of the quotient of coefficient of
error (CE = SEM/mean) and CV [CE2/CV2] should be below 0.5.
Values in the present study for areas of thalamic nuclei in control
patients were LPN = 0.089, VPN = 0.013, DMN = 0.023; and for
cell counts LPN = 0.018, VPN = 0.024, DMN = 0.0014). Thus, the
precision of the estimate obtained with the sampling scheme used
in this study was greater than that required for optimal sampling
(West et al., 1991). A precise estimate of the total cell number in
a known volume of tissue (West and Gundersen, 1990) was obtained
using the equation NV = Q/V, where NV is the numerical density,
Q is the number of cells counted in each frame, and V is the volume
of the counting frame. The total number of neurons within each
counting box was counted; the mean value was calculated and multiplied by the number of counting boxes present within each thalamic
nucleus. All neurons containing a nucleolus were counted in order to
overcome difficulties in interpretation of the viability or otherwise of
‘dark’ neurons compared with normal staining neurons.
Statistical analysis
The statistical significance of differences in areas of cross-section of
VPN, LPN and DMN in the thalamus of control, moderately disabled, severely disabled and vegetative head-injured patients was
analysed using (i) analysis of variance (ANOVA), within 95% confidence intervals; (ii) the Tukey–Kramer multiple comparisons test;
and (iii) unpaired t tests with a Welch correction for differences
between control and subgroups of head-injured patients. The statistical significance for differences in (i) the mean transverse diameter
of neurons and (ii) the number of neurons within (a) counting boxes
and (b) a 28 mm thick coronal section at the level of the lateral
2474
W. L. Maxwell et al.
geniculate body was calculated using (a) ANOVA and (b) unpaired
t tests with a Welch correction in control and all groups of headinjured patients for each nucleus. Significance was assumed when
P < 0.05.
Results
Changes in cross-sectional area of DMN, VPN
and LPN after blunt head injury
The cross-sectional areas, in mm2, for the thalamic DMN,
VPN and LPN at the level of the lateral geniculate body in
all patients are provided in Table 2. ANOVA showed that the
cross-sectional area for each nucleus differed significantly
between all experimental groups (P < 0.0001). More specifically, use of the Tukey–Kramer multiple comparison test
showed that the cross-sectional area of each nucleus did not
differ between control and the moderately disabled patients
(P > 0.05, q for DMN = 0.607, for VPN = 0.775 and for
LPN = 0.280). However, in the severely disabled patients,
there was a significant loss of cross-sectional area of the
DMN nucleus to 73.3% (P < 0.001, q = 11.91) and in the
vegetative patients to 70.86 % (P < 0.001, q = 12.75) of
control values. There was a significant loss of cross-sectional
area of the VPN only in the vegetative patients (P = 0.04,
q = 3.82). There was no change in the cross-sectional area of
LPN in any patient group with moderate disability, severe
disability or who were vegetative (P = 0.83).
Moreover, comparison using the Bonferoni t test of crosssection area between pairs within and between experimental
groups showed that changes within a particular thalamic
nucleus varied with the GOS in that there was a highly significant loss from the DMN in moderately disabled, severely
disabled head-injured (P = 0.0007, t = 4.30) and vegetative
(P = 0.0003, t = 4.71) patients. On the contrary, there was not
a significant difference between the control and vegetative
patients (P = 0.06, t = 2.04) for VPN or for LPN (P = 0.373)
(Table 2).
Changes in mean neuronal diameter
There was a significant difference in the size of neurons
between the DMN, VPN and LPN in control patients. Within
the DMN, the median cell diameter was 7.4 6 0.19 mm, within
VPN the value was 11.3 6 0.70 mm, and within LPN the value
was 14.4 6 1.14 mm. ANOVA demonstrated a significant
difference between groups (P < 0.0001). After head injury
and survival in a vegetative state for >3 months, morphological change comparable with that described in other studies
(reviewed in Ross and Ebner, 1990; Xuereb et al., 1991) was
noted in neurons. Neuronal cell bodies were either within the
control size range with a well-defined, pale central nucleus or
reduced in size with a darkened cytoplasm, a peripherally
located nucleus and the cell often lying at the edge of its
residual lacuna within the neuropil. However, analysis of
differences in mean neuronal diameter did not support the
hypothesis that there was an overall change in neuronal
soma dimensions for either DMN (P = 0.88) or VPN (P =
0.076). This is due to the fact that the proportion of small, dark
neurons did not differ between control and head-injured
patients. However, there was a significant change in the
mean size of neurons within the LPN (P < 0.0001, t =
7.12). Thus, in long-term vegetative patients, there is an
ongoing response by neurons in LPN of patients who survive
for >3 months.
Number of neurons within the DMN, VPN and
LPN of the thalamus in control and
head-injured patients
Comparison of the number of neurons in counting boxes
within each nucleus between control and head-injured
patients using ANOVA showed no significant differences
(Table 2). Comparison of pairs between control and each
of the head-injured groups using the Bonferroni t test further
demonstrated that there was no difference in the number of
neurons occurring within a standardized counting box within
DMN, VPN and LPN. The P values were: P = 0.99 (not
significant) for the DMN; P = 0.99 (not significant) for the
VPN; and P = 0.98 (not significant) for the LPN.
Data obtained from randomly selected fields within each of
the thalamic nuclei did not support the hypothesis that there
is loss of neurons from the DMN, VPN or LPN with either
survival or the category of outcome after head injury.
However, when the entire cross-sectional area of the DMN
and LPN with survival was analysed, a different result was
obtained. Counts for the total number of neurons within a
28 mm thick slice at the level of the lateral geniculate body
in controls, moderately and severely disabled, or vegetative
patients after TBI, as assessed by the GOS, demonstrated
differences between both patient groups and the severity of
injury. Overall, there was loss of neurons from the DMN
(ANOVA, P = 0.03) and VPN (ANOVA, P = 0.0415), but
no loss from the LPN (ANOVA, P = 0.055). The patient
groups examined in this present study provide novel information in support of the hypothesis that neuronal loss occurred in
the DMN of moderately disabled patients (P = 0.04, t = 2.14)
(Table 2). However, no evidence for increased numbers of
small, dark neurons, possibly undergoing degeneration (Ross
and Ebner, 1990), was obtained. Analysis of pairs of patient
groups showed some novel and interesting differences
between patients within the severely disabled and vegetative
groups. In the DMN, there was a significant loss of neurons
both in severely disabled (P < 0.0001, t = 9.24) and in vegetative patients (P < 0.0001, t = 10.356). Similarly, in the VPN,
there was, again, a significant loss of neurons in both severely
disabled (P < 0.0001, t = 7.188) and vegetative (P < 0.0001,
t = 7.96) patients (Table 2). However, for the LPN, there was
no difference (P = 0.76) overall or between pairs (P = 0.55).
A hypothetical explanation for this result is that degeneration of neurons in the DMN and VPN occurs rapidly after head
Thalamic responses after human blunt head injury
injury and there is no significant change thereafter (see
Discussion). A different result, however, was obtained for
the LPN. The cross-sectional area of the LPN is unchanged
between control and all categories of patients. Nor is there any
evidence for loss of neurons obtained in that the total number
did not differ (P = 0.37) between these groups. However, there
was a significant overall reduction in the size of the neurons
(unpaired t test with Welch correction P < 0.0001, t = 7.12)
between control and head-injured patients within the LPN. It
may be posited, therefore, that in long-term survival vegetative patients, there is a low grade, ongoing loss of neurons
from the LPN.
Discussion
The present study provides the first quantitative evidence for
neuronal loss from nuclei within the thalamus of moderately
disabled, severely disabled and vegetative head-injured
patients as assessed by the GOS (Jennet and Bond, 1975).
Our results provide novel evidence for (i) different rates of
loss of neurons between thalamic nuclei and (ii) for quantitative evidence of an ongoing response by neurons within the
LPN when post-traumatic survival is >4 months in both the
severely disabled and vegetative patients.
Three thalamic nuclei have been investigated in the present
study. The fact that there was no change in the cross-sectional
area of the LPN indicates that the changes in area for the DMN
and VPN discussed below are not the result of artefact.
Increased confidence in this observation is provided by the
fact that sites of neuronal degeneration were anatomically
discrete. This finding is in parallel with the findings of Xuereb
et al. (1991) in Alzheimer’s patients in which nuclear volume/
cell loss occurred only in the anterodorsal thalamus but not
the centromedian nucleus. The present study provides the first
quantitative evidence for loss of neurons within thalamic
nuclei after TBI. However, importantly, such changes
occurred both to different proportions of the total number
of neurons within each nucleus and at different levels of
patient category as assessed by the GOS.
Brain atrophy, measured as a significant change in volume,
has been documented in the putamen/globus pallidus and the
hippocampus (Cullum and Bigler, 1986; Anderson and Bigler,
1995; Tate and Bigler, 2000) of patients that have survived
TBI. However, in vivo imaging techniques, at present, cannot
provide data for changes, for example, in the number of neurons. Loss of brain tissue has also been documented in several
experimental studies of TBI. However, changes in brain structure and relationships have been noted largely in terms of
atrophy of the cerebrum at the site of impact and hydrocephalus of the ipsilateral, lateral ventricle (Bramlett et al.,
1997; Smith et al., 1997; Pierce et al., 1998; Dixon et al.,
1999). There are also differences in outcome between the
experimental model used, for example fluid percussion injury,
compared with the loading conditions acting during human
blunt head injury. For example, cortical atrophy and communication between the lateral ventricle and a cystic lesion at
2475
the site of impact has not been systematically documented
in human TBI. This may largely be because, in human TBI,
direct impact to the exposed surface of the meninges occurs
rarely. Thus, as far as we are aware, a stereological analysis
for loss of a specific type of cell has not yet been undertaken
in any model of TBI.
Experimental models may allow an appraisal of changes in
post-traumatic behaviour and learning functions (Smith et al.,
1997; Pierce et al., 1998; Dixon et al., 1999), but only at a
relatively simplistic level. It is known that in man the DMN
has reciprocal connections between the prefrontal, orbital and
temporal cortices, connections to the amygdala and the limbic
system, and other thalamic nuclei and association areas of the
parietal and temporal lobes. These are involved with learning,
interpretation of written symbols and generation of speech.
Clearly, information with regard to deterioration in language,
cognitive function and social interaction is difficult to obtain
in animal models. Neuropsychological data in moderately
disabled patients have documented a significant loss for
IQ scores, for consistent retrieval from long-term storage memory and for slowed or perturbed thinking (Levin et al., 1979).
More recently, Levine et al. (2002) have reported reduced
activity in the dorsomedial thalamus in memory retrieval
using PET scans from patients that had experienced moderate
or severe TBI 4 years before scanning. Evidence has also
been obtained for quantitative changes in blood flow and
glucose utilization both in humans in either epilepsy (Juhasz
et al., 1999) or TBI (Stamatakis et al., 2002), and in animal
models of TBI (Moore et al., 2000). Also there is strong
evidence for recovery of cognitive function despite a reduction in volume of the brain during recovery (Johnson et al.,
1994). Patients within the present study were from a different
population from that used by both Levin et al. (1979) and
Johnson et al. (1994). Nonetheless, it is intriguing that, in the
present study, a significant loss of neurons had occurred from
the DMN in the moderately disabled patients (mean survival
7 years). Difficulties with language, behaviour, social interaction or rational thinking are frequent in moderately and
severely disabled head-injured patients, but an integrated
study using in vivo imaging techniques and neuropsychological analyses is required to further our understanding in
this complex field.
The present study provides novel, quantitative evidence
that there is not a single time course for loss of neurons
from the human thalamus after TBI. The lack of change in
neuronal size together with the significant loss in crosssectional area of the DMN and VPN observed in the present
study is posited to be evidence for a rapid loss of neurons from
these nuclei post-trauma. For example, after experimental
cortical ablation, shrunken dark cells occur only between
7 and 14 days in the thalamic ventrobasal complex, lateral
geniculate nuclei and VPN in larger experimental animals
such as cat or monkey (Carreras et al., 1969; Wong-Riley,
1972) and up to 14 days after fluid percussion injury in the rat
(Conti et al., 1998). In addition, not all neurons within
the thalamus degenerate where persisting cells have been
2476
W. L. Maxwell et al.
suggested to be interneurons (Ross and Ebner, 1990). In the
present study, the patient with the shortest survival died
4 weeks after TBI, a period probably longer than that required
for post-traumatic neuronal degeneration. It is suggested that
in the DMN and VPN of humans after blunt head injury, many
neurons degenerate within 3–4 weeks of injury and that there
is no further significant loss of neurons thereafter. At a very
basic level, the degree of cell loss between nuclei varies with
the severity of the outcome. In moderately disabled patients,
10% of neurons are lost in the DMN but only 5% in both
the LPN and VPN. In severely disabled patients, there was
loss of 20% of neurons from the DMN and 16% from the
VPN. Thus, there is a greater loss from the DMN. This trend
was maintained in vegetative patients where the loss of neurons in the DMN was >30% while in the VPN it was <20%.
Although such loss of neurons was comparable with the percentage loss in cross-sectional area of the DMN and VPN in
non-human primates, it is not yet possible to form a direct
correlation. The experimental literature (Ross and Ebner,
1990) indicates that glial cells within the thalamus also
respond after TBI. Quantitative data for alterations in the
number of glial cells within the thalamus after human blunt
head injury presently are lacking. Nonetheless, the following
observations may be made although it is recognized that the
patient population is relatively small and there is some heterogeneity within the pathological findings. After blunt head
injury, the greatest loss of neurons was from the DMN in
all three categories of patient assessed by the GOS. There
is a lesser loss from the VPN, with statistical significance
only being obtained in severely disabled and vegetative
patients. Stereology did not support the hypothesis that
there was either post-traumatic loss of neurons or a change
in the cross-sectional area of the LPN. Nevertheless, uniquely
between the three nuclei examined, there was a significant
change in the size of neurons within the LPN. Indeed, microscopic examination indicated an increased proportion of
shrunken, dark neurons that might be indicative of neuronal
degeneration. It is suggested that the time course of neuronal
responses after TBI is different between the thalamic LPN and
DMN/VPN.
To our knowledge, only two studies in non-human primates
have analysed the numbers of neurons within thalamic nuclei.
Chmielowska and Pons (1995) reported loss of cytochrome
oxidase-immunolabelled and Nissl-stained neurons in the
VPN after damage to the postcentral somatosensory cortex
in monkeys. Xuereb et al. (1991) reported loss of neurons
from the anterodorsal thalamic nuclei from patients with
Alzheimer’s disease and Parkinson’s disease using cresyl
violet and Luxol fast blue staining. Direct comparison for
humans is not possible since Xuereb et al. (1991) did not
provide details of the ages of patients in their analysis.
Nonetheless, for patients with Alzheimer’s disease, there was
a 27% reduction in the total volume of the dorsal thalamus. In
the present study, a comparable loss, 26.6% in severely disabled patients and 29.1% in vegetative patients, in the DMN
was obtained. Further, the present data allow development of
the hypothesis that such loss occurs within 4 weeks after
injury. However, it is noted that assessment by GOS is not
undertaken until 6 months post-insult to allow stabilization of
the patient’s clinical condition.
The present study provides the first quantitative evidence
for loss of neurons from the VPN after blunt head injury. The
VPN receives input from the extremities, trunk and face and
projects to the postcentral gyrus (Jones, 1985). Transverse
sections of the brainstem of severely disabled and vegetative
patients show degeneration of afferent and efferent pathways
(Strich, 1956; Adams et al., 2000). Within the thalamus, axons
in the dorsal columns terminate on large, parvalbuminpositive neurons, while axons in the spinothalamic pathway
terminate on smaller, calbindin-positive neurons. However,
these are just two among other groups of neurons (cytochrome
oxidase-positive and GABA positive) lost after somatosensory cortical ablation (Rausell et al., 1992; Chmielowska
and Pons, 1995). Thus, only a fraction of the total number of
neurons in the VPN will be de-afferented following head
injury and undergo degeneration.
Correspondingly, only a relatively small percentage of neurons from the VPN in head-injured patients might be expected
to degenerate. In the present study, between 16 and 18% of
neurons were lost from the VPL nucleus in severely disabled
and vegetative patients compared with 20–30% from the
DMN in the same patients. These differences provide
good, quantitative evidence that responses differ between
different thalamic nuclei after TBI. However, our present
understanding of the thalamocortical projections from the
VPN is incomplete and the different types of neurons within
that nucleus greatly complicate research in this area. In simple
terms, Rausell et al., (1992) indicate that some calbindin
neurons project to superficial layers of more than one postcentral cortical area. At present, quantitative data for neuronal
loss in human cerebral cortex, hypothetically due to transneuronal degeneration after TBI, are lacking and definitive evidence will require use of a panel of immunocytochemical
markers. It is established that the medial lemniscus and spinothalamic pathways are major afferent sensory tracts. The
novel observation that 16–18% of thalamic relay neurons
were lost in the present study may suggest compromised
sensory responses following TBI.
Cellular mechanisms for neuronal loss post-trauma were
not a remit of the present study. Review of the literature
indicates that knowledge of the detailed intercellular relationships of human thalamic neurons is still incomplete. However,
the present study does provide quantitative evidence that there
is, at least, more than a single mechanism and time course for
neuronal loss and that the degree of loss differs between both
thalamic nuclei and different categories of patients assessed
by the GOS. Further, there are many types of interneuron
within the human thalamus (reviewed Ross and Ebner,
1990; Rausell et al., 1992; Chmielowska and Pons, 1995).
A mechanistic analysis is therefore limited by the present lack
of detailed knowledge concerning thalamic input originating
either from the cerebral cortex or from the brainstem (Ross and
Thalamic responses after human blunt head injury
Ebner, 1990; Rausell et al., 1992; Chmielowska and Pons,
1995). Nonetheless, the present study provides novel, quantitative data that there is, at least, more than one phase of loss
of neurons from the thalamus after human blunt head injury.
For example, rapid loss resulting from either oligaemia
(Stamatakis et al., 2002) and altered glucose metabolism
(Juhasz et al., 1999) or retrograde thalamic degeneration
which in man takes some 3 months post-trauma to develop
(Adams et al., 2000). Further, it is noteworthy that the nuclei
from which loss occurred in the present study (DMN and
VPN) differ from those noted in Alzheimer’s disease (anterodorsal and centromedian) (Xuereb et al., 1991). Further analysis will probably require complex immunocytochemical
analysis of which particular subgroup(s) of neurons within
the human thalamus respond and/or are lost after TBI.
In conclusion, overall, it must be acknowledged that the
GOS is a clinical assessment at the level of whole brain
function in the head-injured patient. The present study provides information only for changes in the number of neurons
within specific nuclei with survival or different levels of
severity of head injury, as judged by the GOS, after TBI.
However, such alterations in the total post-traumatic population of neurons may dramatically alter the outcome for the
head-injured patient or the circumstance of that individual’s
family. An earlier analysis of differential responses by
hippocampal neurons (Maxwell et al., 2003) has provided
evidence for an ongoing, long-term post-traumatic response
within one functionally important region of the brain posttrauma. The present study provides quantitative evidence that
further comparative analysis of neuronal, and glial, responses
occurring in other regions of the brain of patients that survive
blunt head injury may be informative with regard to the
pathology influencing the outcome of neurons and glial
cells that may be susceptible to cell death after human
blunt head injury.
Patients for inclusion within this study were assessed using
the GOS. The GOS takes into account overall social capacity
or disability of the patient to provide a measure as to how
independent is the patient to be able to return to normal social
life. Both mental and physical disabilities contribute to the
GOS (Jennett, 1997). The GOS is not a specific measure of
loss of specific brain functions. The correlation between
thalamic injury and category of GOS, obtained in this
study, is very encouraging. However, it cannot be absolute
because neuronal loss or other cellular changes in other brain
regions will contribute to any result obtained using the GOS.
Further, additional structural changes may occur as a result of
post-traumatic epilepsy, substance (alcohol) abuse and other
aspects of lifestyle especially for the moderately disabled
group (Adams et al., 2001). Additionally, clinical studies
suggest that TBI may accelerate or exacerbate cognitive
decline associated with normal ageing (Stuss et al., 1989)
and induce Alzheimer’s disease-like neurodegeneration
(Mortimer et al., 1991; Gentleman et al., 1993; Mayeux
et al., 1993; Nicoll et al., 1995; Geddes et al., 1996). Analyses
of changes in neuronal and glial numbers in other brain areas
2477
are being undertaken in the cases used in this study to provide
a more detailed picture of the structural basis of the GOS.
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