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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). 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