Detecting residual cognitive function in disorders of consciousness.

Clinical audits have suggested up to 40% of patients with disorders of consciousness may be misdiagnosed, in part, due to the highly subjective process of determining, from a patient's behaviour, whether they retain awareness of self or environment. To address this problem, objective neuroimaging methods, such as positron emission tomography and functional magnetic resonance imaging have been explored. Using these techniques, paradigms, which do not require the patient to move or speak, can be used to determine a patient's level of residual cognitive function. Indeed, visual discrimination, speech comprehension and even the ability to respond to command have been demonstrated in some patients who are assumed to be vegetative on the basis of standard behavioural assessments. Functional neuroimaging is now increasingly considered to be a very useful and necessary addition to the clinical assessment process, where there is concern about the accuracy of the diagnosis and the possibility that residual cognitive function has remained undetected. In this essay, the latest neuroimaging findings are reviewed, the limitations and caveats pertaining to interpretation are outlined and the necessary developments, before neuroimaging becomes a standard component of the clinical assessment are discussed.

A multimodal approach to the assessment of patients with disorders of consciousness.

Unlike other neurological conditions, the heterogeneous pathology linked to disorders of consciousness currently excludes a distinction between the vegetative and minimally conscious states based upon pathological presentation. The clinical assessment is therefore made on the basis of the patient's clinical history and exhibited behaviour. This creates a particular challenge for the clinician who has to decide whether a certain behaviour, which might be inconsistent or incomplete, reflects a conscious or an unconscious process. In an alarmingly high number of cases, identified during clinical audit, this decision process has been shown to be particularly fallible. The behavioural assessment is not only highly subjective, but also dependent upon the ability of the patient to move or speak; it is the only way someone can demonstrate they are aware. To address this problem we propose a multimodal approach, which integrates objective tools, such as electrophysiology and functional brain imaging, with traditional behavioural scales. Together this approach informs the clinical decision process and resolves many of the dilemmas faced by clinicians interpreting solely behavioural indices. This approach not only provides objective information regarding the integrity of residual cognitive function, but also removes the dependency on the patient to move or speak by using specially designed paradigms that do not require a motor output in order to reveal awareness of self or environment. To demonstrate this approach we describe the case of BW, who sustained a traumatic brain injury seven months prior to investigation. BW was admitted to a five-day assessment programme, which implemented our multimodal approach. On behavioural assessment BW demonstrated evidence of orientation and visual pursuit. However, he showed no response to written or verbal command, despite holding command cards and scanning text. Electrophysiology confirmed that he retained a preserved neural axis supporting vision and hearing, and suggested some evidence that he was able to create a basic memory trace. A hierarchical fMRI auditory paradigm suggested he was able to perceive sound and speech, but revealed no evidence of speech comprehension or ability to respond to command. This was corroborated in the visual modality using a hierarchical paradigm demonstrating that he was able to perceive motion, objects and faces, but retained no evidence of being able to respond to command. We briefly review work by other teams advocating the use of brain imaging and electrophysiology and discuss the steps that are now required in order to create an international standard for the assessment of persons with impaired consciousness after brain injury.

Towards the routine use of brain imaging to aid the clinical diagnosis of disorders of consciousness.

Clinical audits have highlighted the many challenges and dilemmas faced by clinicians assessing persons with disorders of consciousness (vegetative state and minimally conscious state). The diagnostic decision-making process is highly subjective, dependent upon the skills of the examiner and invariably dictated by the patients' ability to move or speak. Whilst a considerable amount has been learnt since Jennett and Plum coined the term 'vegetative state', the assessment process remains largely unchanged; conducted at the bedside, using behavioural assessment tools, which are susceptible to environmental and physiological factors. This has created a situation where the rate of misdiagnosis is unacceptably high (up to 43%). In order to address these problems, various functional brain imaging paradigms, which do not rely upon the patient's ability to move or speak, have been proposed as a source of additional information to inform the diagnostic decision making process. Although accumulated evidence from brain imaging, particularly functional magnetic resonance imaging (fMRI), has been encouraging, the empirical evidence is still based on relatively small numbers of patients. It remains unclear whether brain imaging is capable of informing the diagnosis beyond the behavioural assessment and whether brain imaging has any prognostic utility. In this study, we describe the functional brain imaging findings from a group of 41 patients with disorders of consciousness, who undertook a hierarchical speech processing task. We found, contrary to the clinical impression of a specialist team using behavioural assessment tools, that two patients referred to the study with a diagnosis of vegetative state did in fact demonstrate neural correlates of speech comprehension when assessed using functional brain imaging. These fMRI findings were found to have no association with the patient's behavioural presentation at the time of investigation and thus provided additional diagnostic information beyond the traditional clinical assessment. Notably, the utility of brain imaging was further underlined by the finding that the level of auditory processing revealed by functional brain imaging, correlated strongly (rs = 0.81, P < 0.001) with the patient's subsequent behavioural recovery, 6 months after the scan, suggesting that brain imaging may also provide valuable prognostic information. Although further evidence is required before consensus statements can be made regarding the use of brain imaging in clinical decision making for disorders of consciousness, the results from this study clearly highlight the potential of imaging to inform the diagnostic decision-making process for persons with disorders of consciousness.

Circadian rhythms in the vegetative state.

OBJECTIVE: To evaluate whether vegetative state patients maintain circadian rhythms. RESEARCH DESIGN: An observational study of five single cases. METHODS AND PROCEDURES: Five chronic vegetative state patients underwent clinical and neurological evaluations and 2-week continuous temperature measurements. MAIN OUTCOMES AND RESULTS: The two patients with traumatic brain injury showed well-formed circadian temperature rhythms and had more reflexive behaviours and relatively low cortical and sub-cortical atrophy, whereas the three patients from anoxic-hypoxic origin demonstrated no cycles or rhythmic behaviour. CONCLUSIONS: The presence of periods of wakefulness does not imply preserved sleep-wake cycling capacity, nor preserved circadian rhythms and it should not be taken as a distinguishing feature for the definition of the vegetative state.

Can electromyography objectively detect voluntary movement in disorders of consciousness?

Determining conscious processing in unresponsive patients relies on subjective behavioural assessment. Using data from hand electromyography, the authors studied the occurrence of subthreshold muscle activity in response to verbal command, as an objective indicator of awareness in 10 disorders of consciousness patients. One out of eight vegetative state patients and both minimally conscious patients (n = 2) demonstrated an increased electromyography signal specifically linked to command. These findings suggest electromyography could be used to assess awareness objectively in pathologies of consciousness.

When thoughts become action: an fMRI paradigm to study volitional brain activity in non-communicative brain injured patients.

The assessment of voluntary behavior in non-communicative brain injured patients is often challenging due to the existence of profound motor impairment. In the absence of a full understanding of the neural correlates of consciousness, even a normal activation in response to passive sensory stimulation cannot be considered as proof of the presence of awareness in these patients. In contrast, predicted activation in response to the instruction to perform a mental imagery task would provide evidence of voluntary task-dependent brain activity, and hence of consciousness, in non-communicative patients. However, no data yet exist to indicate which imagery instructions would yield reliable single subject activation. The aim of the present study was to establish such a paradigm in healthy volunteers. Two exploratory experiments evaluated the reproducibility of individual brain activation elicited by four distinct mental imagery tasks. The two most robust mental imagery tasks were found to be spatial navigation and motor imagery. In a third experiment, where these two tasks were directly compared, differentiation of each task from one another and from rest periods was assessed blindly using a priori criteria and was correct for every volunteer. The spatial navigation and motor imagery tasks described here permit the identification of volitional brain activation at the single subject level, without a motor response. Volunteer as well as patient data [Owen, A.M., Coleman, M.R., Boly, M., Davis, M.H., Laureys, S., Pickard J.D., 2006. Detecting awareness in the vegetative state. Science 313, 1402] strongly suggest that this paradigm may provide a method for assessing the presence of volitional brain activity, and thus of consciousness, in non-communicative brain-injured patients.

Monitoring emergence from coma following severe brain injury in an octogenarian using behavioural indicators, electrophysiological measures and metabolic studies: a demonstration of the potential for good recovery in older adults.

This case study describes a multi-disciplinary investigation of the emergence from coma of an 80-year old female (KE) following severe traumatic brain injury. The relationship between cognitive/behavioural ability and the integrity of cerebral function was assessed using neuropsychological measures, positron emission tomography, electroencephalography, somatosensory evoked potentials and trans-cranial magnetic stimulation. These investigations were performed as KE was beginning to emerge from coma (4 weeks) and, again, approximately 1 year following brain injury, when she was judged to have achieved her maximum level of recovery. Neuropsychological measures revealed improvement during the first year post-injury in KE's speed of information processing, memory and executive abilities. Electrophysiological and metabolic studies indicated a restoration of functional integrity that was consistent with the gradual recovery in higher brain function documented using behavioural procedures. This case study demonstrates the rehabilitation potential of pre-morbidly healthy older adults following severe traumatic brain injury.

Neurometabolic coupling in the vegetative and minimally conscious states: preliminary findings.

The objective of this study was to determine the integrity of the homoeostatic coupling relationship between neuronal electrical function and cerebral metabolism in the vegetative and minimally conscious states. Ten patients who met recognised diagnostic criteria (six in the vegetative state and four minimally conscious) were investigated using simultaneous electroencephalography and positron emission tomography. It was found that the coupling between neuronal electrical activity and regional glucose metabolism was preserved in all the minimally conscious patients but was absent in all the vegetative state patients. Our preliminary results suggest patients in the vegetative state may endure an impaired coupling relation between neuronal electrical function and cerebral energy metabolism.

Imaging of cerebral blood flow and metabolism in brain injury in the ICU.

The heterogeneity of the initial insult and subsequent pathophysiology has made both the study of human head injury and design of randomised controlled trials exceptionally difficult. The combination of multimodality bedside monitoring and functional brain imaging positron emission tomography (PET) and magnetic resonance (MR), incorporated within a Neurosciences Critical Care Unit, provides the resource required to study critically ill patients after brain injury from initial ictus through recovery from coma and rehabilitation to final outcome. Methods to define cerebral ischemia in the context of altered cerebral oxidative metabolism have been developed, traditional therapies for intracranial hypertension re-evaluated and bedside monitors cross-validated. New modelling and analytical approaches have been developed.

Effects of propofol on cerebral oxygenation and metabolism after head injury.

BACKGROUND: Flow-metabolism coupling is thought to be deranged after traumatic brain injury, while the effects of propofol on flow-metabolism coupling are controversial. We have used a step increase in target plasma propofol concentration in head injured patients to explore flow-metabolism coupling in these patients. METHODS: Ten patients with a moderate to severe head injury received a step increase in propofol target controlled infusion of 2 microg x ml(-1). Cerebral tissue gas measurements were recorded using a multimodal sensor, and regional chemistry was assessed using microdialysis. Arterial-jugular venous oxygen differences (AVDO(2)) were measured and all patients had cortical function monitoring (EEG). RESULTS: The step increase in propofol led to a large increase in EEG burst-suppression ratio (0% (range 0-1.1) to 46.1% (range 0-61.7), P<0.05); however, this did not significantly change tissue gas levels, tissue chemistry, or AVDO(2). CONCLUSIONS: Flow-metabolism coupling remains intact during a step increase in propofol after traumatic brain injury. The EEG burst-suppression induced by propofol after traumatic brain injury does not appear to be a useful therapeutic tool in reducing the level of regional ischaemic burden.

Investigation of the effect of chlormethiazole on cerebral chemistry in neurosurgical patients: a combined study of microdialysis and a neuroprotective agent.

AIMS: Promising pre-clinical results from laboratory studies of neuro-protective drugs for the treatment of patients with stroke and head injury have not been translated into benefit during clinical trials. The objective of the study was to assess the feasibility of administrating a potential neuro-protective drug (chlormethiazole) in conjunction with multimodality monitoring (including microdialysis) to patients with severe head injury in order to determine the effect of the agent on surrogate endpoints and penetration into the brain. METHODS: Multimodality monitoring including cerebral and peripheral microdialysis was applied to five head-injured patients on the neuro-intensive care unit. Chlormethiazole (0.8%) was administered as a rapid (10 ml min(-1)) intravenous loading infusion for 5 min followed by a slow (1 ml min(-1)) continuous infusion for 60 min. The following parameters were monitored: heart rate, mean arterial blood pressure, intracranial pressure, cerebral perfusion pressure, peripheral oxygen saturation, continuous arterial oxygen partial pressure, arterial carbon dioxide partial pressure, arterial pH, arterial temperature, cerebral tissue oxygen pressure, cerebral tissue carbon dioxide pressure, cerebral pH, cerebral temperature, electroencephalograph (EEG), bi-spectral index, plasma glucose, plasma chlormethiazole, and cerebral and peripheral microdialysis assay for chlormethiazole, glucose, lactate, pyruvate and amino acids. RESULTS: Despite achieving adequate plasma concentrations, chlormethiazole was not detected in the peripheral or cerebral microdialysis samples. The drug was well tolerated and did not induce hypotension, hyperglycaemia or withdrawal seizures. The drug did not change the values of the physiological or chemical parameters including levels of GABA, lactate/pyruvate ratio and glutamate. The drug did, however, induce EEG changes, including burst suppression in two patients. CONCLUSIONS: Chlormethiazole can be safely given to ventilated patients with severe head injury. There was no evidence of hypotension or withdrawal seizures. Combining a pilot clinical study of a neuro-protective agent with multimodality monitoring is feasible and, despite the lack of effect on physiological and chemical parameters in this study, may be a useful adjunct to the development of neuro-protective drugs in the future. Further investigation of the capability of microdialysis in this setting is required. By investigating the effect of a drug on surrogate end-points, it may be possible to identify promising agents from small pilot clinical studies before embarking on large phase III clinical trials.

Liquid chromatographic determination of nicarbazin in feeds.

A new liquid chromatographic method has been developed for determination of nicarbazin in feeds. Approximately 40 g feed is extracted with 200 mL acetonitrile-water (80 + 20, v/v). An aliquot of the extract is filtered and assayed using a reversed-phase isocratic method that measures the 4,4'-dinitrocarbanilide moiety of nicarbazin at a wavelength of 340 nm. For medicated feeds, the method uses a standard linear range of 5 to 100 microg/mL. For lower levels, a linear range of 50 to 150 ng/mL can be used. The method has a limit of detection of 250 ng/g and a limit of quantitation of 500 ng/g in a 40 g feed sample. Recovery was 99.1%, with a range of 95.2 to 101.8%. In the typical U.S. dosing range of 27 to 113.5 g/ton, the precision of the method based on one analyst, one day, and 2 weighings ranged from 2.8% (113.5 g/ton) to 4.7% (27 g/ton).

Determination of avilamycin in poultry feeds by liquid chromatography.

Avilamycin was extracted from feed with acetonitrile. Isolation of avilamycin factors from feed matrix interference was accomplished by normal-phase solid-phase extraction with silica as sorbent. Reversed-phase liquid chromatography was subsequently used to separate and quantitate the primary biologically active factors A and B for determination of chemical potency. This method combines specificity for avilamycins A and B in poultry feeds with simple sample preparation that removes matrix interferences. Recoveries of factor A ranged from 93.29 to 97.26%, with precision (relative standard deviation) ranging from 1.1 to 3.4%. Avilamycin factors in feed samples tested ranged from 4.45 to 17.82 micrograms/g for factor A and from 0.80 to 3.18 micrograms/g for factor B.

Determination of apramycin in swine kidney tissue by liquid chromatography with fluorescence detection.

A liquid chromatographic method for determining apramycin in swine kidney tissue is described. Apramycin is extracted from tissue with basic methanol and purified by ion-pair extraction. By using an automated derivatization and injection procedure, the purified extract is derivatized with o-phthaldehyde, separated on a C18 column, and detected with a fluorescence detector. For fortified kidney samples, between-run coefficients of variation ranged from 4.8 to 7.1% at 1.00 ppm and from 9.6 to 14.3% at 0.50 ppm. Recoveries ranged from 76 to 86%. Standard curves were linear over the range 10-100 ng/mL.

Liquid chromatographic determination of monensin in premix and animal feeds: collaborative study.

An interlaboratory study of a liquid chromatographic (LC) method for determining monensin in premix (60-80 g/lb or 132-176 mg/g) and animal feeds (5-200 g/ton or 0.0055-0.22 mg/g) was conducted in laboratoriesin the United States, Canada, France, and Germany. The LC system used a reversed-phase column, postcolumn derivatization with vanillin, and UV detection. The method separates monensin from other ionophores such as narasin and salinomycin. Each laboratory analyzed a total of 20 samples of premix, liquid feed supplements, poultry, and cattle feeds. Concentrations of monensin in all samples ranged from 0 to 176 mg/g (80 g/lb). Reproducibility relative standard deviation (RSDR) for premix ranged from 2.8 to 3.4%. For feed samples containing monensin, repeatability standard deviation (sr) ranged from 0.9 to 7.0. Reproducibility standard deviation (sR) ranged from 1.2 to 11. Repeatability relative standard deviation (RSDr) ranged from 6.1 to 21% and RSDR values-ranged from 8.6 to 25%. Sample preparation for the LC method is less labor intensive than that for the microbiological assays. The LC assay is more efficient than the microbiological assays. This LC method for determination of monensin in premix and animal feeds has been adopted first action by AOAC INTERNATIONAL.

Liquid chromatographic determination of tilmicosin in swine feed at 200-400 mg/kg level: interlaboratory study.

An analytical method for the determination of tilmicosin at 200-400 mg/kg, the intended use concentration range, was evaluated in an interlaboratory study involving 5 laboratories, including the sponsor. The interlaboratory study evaluated the intra- and interlaboratory precision and accuracy of a tilmicosin feed method. The method procedure involved extracting tilmicosin from feed by adding 200 mL extractant to 20 g feed and shaking for 1 h. The extract is filtered and analyzed by gradient liquid chromatography which separates tilmicosin from feed matrix in 30 min. Each laboratory assayed 5 replicates of fortified feed at concentrations of 0, 100, 200, 400, and 600 mg/kg. The mean recovery among fortified samples ranged from 81.4 to 98.8%, with a percent coefficient of variation (%CV) ranging from 0.3 to 4.0%. For all blank control feed samples no significant interferences were observed. In addition, each laboratory assayed 5 replicates of medicated feed samples prepared at 2 levels (200 and 400 mg/kg) with either a horizontal or vertical mixer. Along with the medicated feed samples were included 5 replicates of a blank control feed. The identities of the medicated and blank control feed samples were blinded to the analysts. The results for the medicated feed samples ranged from 95.8 to 106% of label claim, with a %CV ranging from 2.1 to 6.7%.

Determination of tilmicosin in bovine and porcine sera by liquid chromatography.

A liquid chromatographic (LC) assay is described for determining tilmicosin in bovine and porcine blood sera. Tilmicosin is isolated from the serum matrix and purified by solid-phase extraction with C18 sorbent. Sample is analyzed by LC using a gradient system with a phenyl reversed-phase column that separates tilmicosin from the matrix in 30 min. Tilmicosin is measured by UV absorbance at 280 nm. Validation of assay included evaluation of accuracy, precision, linearity, specificity, sensitivity, range, and sample stability. The method has a limit of quantitation of 0.1 ppm and a validated range of 0.1 to 10.0 ppm. Recoveries were 91-95% for bovine serum and 85-93% porcine serum. The limit of detection was 0.05 microgram/mL. Limits of detection and quantitation were based on 3 and 6 times the baseline noise of control serum samples, respectively. Relative standard deviations of precision samples (n = 6) were 2% or less for both sera. The method has better specificity and analysis time than previous microbiological methods for tilmicosin in sera.

Determination of monensin in high-moisture cattle rations by liquid chromatography with postcolumn derivatization.

An existing liquid chromatographic method using postcolumn derivatization has been used extensively to quantitate monensin in animal feeds. Because of the relatively high moisture content of many cattle feed rations, some modifications were made to this method. Several sample-processing steps were evaluated to determine optimum sample-processing procedure. The sample weight/sample diluent ratio was modified, and method linearity was validated for the lower monensin concentrations anticipated in high-moisture cattle rations. The accuracy and precision of data generated at these lower concentrations were also determined. Because of the high moisture content of these rations, data analysis for this method required correction of feed potency for loss on drying. With these modifications, monensin can be accurately determined in high-moisture cattle rations.

Determination of ractopamine in monkey plasma and swine serum by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic (HPLC) method is described for the determination of ractopamine (LY031537) in monkey plasma and swine serum. Plasma or serum (0.5 ml) was diluted with phosphate buffer pH 7.0. Ractopamine was isolated from the plasma matrix using ion exchange on a polymeric carboxylic acid solid-phase extraction cartridge followed by partitioning with ethyl acetate. An isocratic HPLC method using electrochemical detection at +700 mV was used to separate and measure ractopamine in the purified extract in 6.5 min of run time. Standard area response was linear with respect to concentration of ractopamine over the range of 0.5 to 40 ng/ml. Validation data were collected using rhesus monkey plasma and swine serum. The method precision and accuracy were evaluated in the range 1.0 to 20 ng/ml using fortified samples of monkey plasma. The method limit of quantitation was estimated at 2 ng/ml as determined in monkey plasma.

Evaluation of ractopamine cross-reactivity in several commercially available beta-agonist enzyme immunoassay kits.

Several enzyme immunoassay test kits are commercially available for screening of matrices including cattle feed, tissues, faeces and urine for the presence of beta-agonists. The kits utilized for this evaluation offer sensitivity as low as 0.1 ng ml-1 (assay solution concentration, not test sample). Evaluation of ractopamine hydrochloride (LY31537) solutions at concentrations as high as 1000 ng ml-1, using six different kits from four different manufacturers, showed cross-reactivity of less than 0.5%.