Tremor in multiple sclerosis

Tremor is estimated to occur in about 25 to 60 percent of patients with multiple sclerosis (MS). This symptom, which can be severely disabling and embarrassing for patients, is difficult to manage. Isoniazid in high doses, carbamazepine, propranolol and gluthetimide have been reported to provide some relief, but published evidence of effectiveness is very limited. Most trials were of small size and of short duration. Cannabinoids appear ineffective. Tremor reduction can be obtained with stereotactic thalamotomy or thalamic stimulation. However, the studies were small and information on long-term functional outcome is scarce. Physiotherapy, tremor reducing orthoses, and limb cooling can achieve some functional improvement. Tremor in MS remains a significant challenge and unmet need, requiring further basic and clinical research.
Search strategy and selection criteria
References for this review were identified from searches of Pubmed (from 1966 to 2005) and the Cochrane Library (2005, Issue 2) with the search terms “multiple sclerosis”, “tremor”, “ataxia”, “disability”, “prevalence”, “surgery”, “thalamotomy”, “deep brain stimulation” and “treatment”. Articles were also identified through searches of the reference tables of identified papers. Furthermore we searched the ISI Science Citation Index for relevant articles citing identified papers. Only articles in English and German were included in the review; publications in abstract form were not considered.
Introduction
Tremor is such a common problem in multiple sclerosis (MS) that the famous French neurologist Charcot (1825–1893) described it as a part of his triad of characteristic symptoms in MS, together with nystagmus, and scanning speech.
Tremor of the upper limbs can be very disabling and seriously impair many activities of daily living and quality of life. In practice the treatment of tremor in MS is often frustrating. In this review we discuss medical, surgical and other treatment options.
Epidemiology
Although the epidemiology of symptoms in MS has been studied extensively, it remains difficult to ascertain the prevalence of tremor in historic study populations, mostly because Kurtzke’s functional systems scale for cerebellar function, which is most widely used in clinical studies in MS, does not record tremor [55]. In a large long-term follow-up study, Weinshenker and colleagues found functionally relevant cerebellar deficits in one third of 259 patients [94]. In another study, disability and dependency of patients with MS were shown to correlate with the severity of ataxia [95].
Two newer studies assessed the prevalence of tremor in MS. One study by Alusi and coworkers examined 100 patients randomly selected from a London MS-clinic and found tremor in 58% of patients. The tremor was minimal in 27%, mild in 16% and moderate to severe in 15% of all patients [6]. As patients were selected from a specialist clinic, the relatively high prevalence and severity of tremor might be due to selection bias.
In a prevalence cohort study by Pittock and colleagues, 200 MS patients living in Olmsted County, Minnesota, USA were assessed for tremor and measures of disability. Tremor was found in 25.5% and severe tremor in 3% of the study population [74]. Probably the community based population in the latter study gives a more realistic estimate of tremor prevalence in MS. In both studies, however, tremor was associated with greater disability as measured on the expanded disability status scale (EDSS). In the Olmsted County population, patients with tremor of any severity were more likely to be unemployed or retired early because of disability.
Tremor subtypes
Tremor in MS can involve the head, neck, vocal cords, trunk and limbs, whereas involvement of the tongue, jaw or palate has not been reported [5].
The different types of tremor are currently classified according to a working consensus of the Movement Disorder Society [29]. In MS, the two most prevalent tremor forms are postural tremor (tremor present whilst voluntarily maintaining a position against gravity) and intention tremor (tremor occurring during target directed movement where tremor amplitude increases during visually guided movements towards the target). True rest tremor (tremor present in a body part that is not voluntarily activated and is completely supported against gravity) is unusual in patients with MS, and Holmes (or “rubral”) tremor is also very uncommon.
In the two main prevalence studies, [6, 74] rest tremor was observed only in the Olmsted County survey (1% of patients), whereas Holmes tremor was observed in neither study. An overview of the affected body parts observed in both studies is given in Table 1. In both studies tremor was most commonly found in the arms. In the London study population, 36% of patients suffered from bilateral arm tremor, making this the most common pattern of limb involvement.
Table 1Details of the two main prevalence studies on tremor in MSLondon [6] (n = 100)Olmsted County [74] (n = 200)Total patients with tremor58 (58%)51 (25.5%)Patients with severe tremor15 (15%)6 (3%)Arm tremor56 (56%)47 (23.5%)Bilateral arm tremor36 (36%)not reportedLeg tremor10 (10%)12 (6%)Head tremor9 (9%)7 (3.5%)trunk tremor7 (7%)not reported
Pathophysiology of tremor
The pathophysiology of tremor in MS is a difficult area of investigation, partly because MS is by definition a multifocal disease, so that tremor occurrence cannot easily be linked to a single neuroanatomical site. Systematic postmortem studies on the link between lesion site and the clinical phenomenon of tremor have never been undertaken.
The predominance of action tremors (postural and intention) in patients with MS point to the cerebellum and its connections as the most likely source of tremor production, whereas the rarity of rest tremor argues against an involvement of the basal ganglia. The common occurrence of bilateral tremor might indicate that damage to the cerebellum and its connections is often multifocal.
Another link to the cerebellum in the pathophysiology of tremor in MS is the effect of peripheral cooling on intention tremor. Intention tremor is thought to be modulated through increased long latency stretch reflexes [30]. Cooling has been shown to decrease the sensitivity of muscle spindles [64] and the velocity of peripheral nerve conduction [56]. In two experimental studies, cooling of the arms markedly reduced intention tremor severity in patients with MS [77, 35]. The authors argue that this effect might have been partly due to decreased muscle spindle function and decreased nerve conduction velocity, which in turn resulted in decreased input into tremor producing cerebellar circuits.
Alusi and coworkers drew attention to the placement site of deep brain stimulation (DBS) electrodes to help understand the neuronal circuits involved in tremor production. They stated that neurosurgeons increasingly chose the nucleus ventralis oralis posterior (VOP) of the thalamus rather than the classic target, the nucleus ventralis intermedius (VIM). This is interesting because the VOP is the basal ganglia output nucleus of the thalamus [6], suggesting that the cerebellar tremors seen in MS might actually be generated by the basal ganglia. The standard electrode placement site reported in the literature, however, is the VIM and no studies comparing placement sites have been published. Whittle and coworkers comment on the difficulty of electrode placement in MS patients: in most patients there are major brain distortions due to demyelination, plaque formation and e vacuo hydrocephalus, and it is therefore uncertain whether the anatomy of these patients conforms to standard stereotactic atlasses [100]. Keeping these comments in mind, it seems unwise to base pathophysiological theories purely on DBS electrode placement site.
A range of animal experiments have been undertaken to indentify the anatomical structures involved in tremor production (for review [101]). It has been shown that damage or removal of the cerebellar cortex does not induce intention tremor in monkeys, [31, 76] whereas partial or complete cerebellectomy leads to tremor during movement and posture [75, 42, 40]. A slow, 3–5 Hz tremor during target directed movements can also be induced by reversible cooling of the dentate nucleus (the origin of most cerebellar efferents) in monkeys [22].
Most cerebellar efferents project from the dentate nucleus via the superior cerebellar peduncle (brachium conjunctivum) to the red nucleus and the thalamus. According to some studies, transection of the superior cerebellar peduncle causes intention tremor in monkeys [34, 93], whereas other authors do not mention this effect.
Interestingly, Carpenter and colleagues report that tremor induced by transection of the superior cerebellar peduncle can be alleviated by a second lesion in the lateroventral or centromedian thalamus. [19, 20] The results of these animal studies suggest that damage to cerebellar efferents (through lesions of the dentate nucleus or superior cerebellar peduncle) may cause disinhibition of thalamic nuclei which are the main producers of intention tremor.
Although this is an interesting pathophysiologic model of intention tremor production, it remains uncertain whether the results of animal studies can be generalized to patients with MS.
In summary, clinical observation, animal studies and some experimental evidence in humans favour the cerebellum and the thalamic nuclei connected to it as the major locus of intention tremor production, but more research is needed to evaluate the role of the basal ganglia and other systems in tremor production in MS.
Assessment of tremor
Depending on the subtype of tremor, several methods for the assessment of tremor severity and its impact on the lives of patients have been developed. Rest tremor is often assessed with the tremor subscale of the Unified Parkinson’s Disease Rating Scale (UPDRS). Fahn and colleagues devised the most comprehensive tremor scale for non-parkinsonian tremor in 1984 [33]. This instrument measures tremor in nine body parts at rest, while maintaining a posture and during goal directed movements. It also includes an assessment of arm tremor while writing and pouring water as well as a subscale for functional disability (interference of tremor with dressing, writing, eating etc.). Some studies included in this review use a modified and heavily abbreviated version of this scale, but most studies assess tremor by clinical examination only (e.g. by finger-to-nose testing, drinking from a cup, nine-hole-peg-test or writing and drawing tasks) or use a simple ordinal severity scale, often classifying tremor as absent, mild, moderate or severe. A simple 0–10 tremor severity scale devised by Bain and coworkers [11] has been shown to be a valid and reliable measurement tool in patients with MS [7], but has so far only been used in few clinical studies [6, 4, 15].
Accelerometry and polarized light goniometry are neurophysiologic methods of tremor assessment. While these methods offer an objective measurement of tremor severity, they can only measure one aspect of an often complex movement problem at a time, and e.g. cannot measure the ataxia which often complicates tremor in MS.
Matsumoto and colleagues devised a more complex “quantitative movement analysis technique”, which records the patients’ goal directed movements in three dimensions using an electromagnetic tracking device, but the complexity of this method [62] as well as the computer aided tracking tasks employed by Aisen and coworkers [2] prohibit their widespread use.
One of the most important aspects of tremor in MS is its impact on the daily life of the patients. Functional status in MS-patients is often measured by asking the patients to complete questionnaires assessing activities of daily living such as writing, eating or dressing. Often these scales are self-devised and not validated. Validated scales used in the studies reviewed in this article include the Frenchay Activities Index [85] and the quality of life subscale of the Functional Assessment of Multiple Sclerosis Scale (FAMS) [62].
Medical treatment
A summary of the published studies on medical treatment of tremor in MS is given in Table 2. Most of the published literature on medical treatment consists of case reports and uncontrolled open label studies. The few randomized controlled trials comprised small numbers of patients and very likely lacked the power to reveal small treatment effects.
Table 2Studies on medical treatment of tremor in MSStudyn*study designintervention(s)tremor assessmentpatients with tremor reduction (%)patients with improved functional status (%)adverse effects (n)Koller, 1984 [52]3double-blind placebo-controlled crossoverpropranolol (not specified) isoniazid (po.) 1200mg/d ethanol (iv.) 50ml of 10% solutionwriting tasks, patient self assessment, clinical examination, accelerometryno effecthepatitis while on isoniazid treatment (1)Sabra et al. 1982 [80]4case reportsisoniazid (po.) 400 to 1200mg/dclinical examination4 (100%)4 (100%)abnormal liver function tests (1)Duquette et al. 1985 [32]12open labelisoniazid (po.) 500–1000mg/dpatient self assessment, clinical examination, blinded evaluation of video tapes10 (83%)0drowsiness, dysphagia, increased bronchial secretions (6) abnormal liver function tests (2) fatigue (2)Morrow et al. 1985 [67]5open labelisoniazid (po.) 700–1200 mg/dclinical examination4 (80%)4 (80%)increased weakness (2) drowsiness (1)Francis et al. 1986 [38]5open labelisoniazid (po.) 1200 mg/dclinical examination, polarised light goniometry4 (80%)0anorexia and nausea (3)Hallet et al. 1985 [44]6double-blind placebo-controlled crossoverisoniazid (po.) 1200 mg/dself-rating scales, accelerometry, blinded evaluation of video tapes6 (100%)0NoneBozek et al. 1987 [16]8double-blind placebo-controlled crossoverisoniazid (po.) 12 or 20 mg/kgclinical examination, accelerometry, blinded evaluation of video tapes6 (75%)4 (50%)somnolence (3) **Sechi et al. 1989 [86]7single-blind placebo controlledcarbamazepine (po.) 400 or 600 mg/dclinical tremor rating scale, accelerometry7 (100%)not reportednone reportedRice et al. 1997 [78]16double-blind placebo-controlled crossoverondansetron (iv.) 8 mg (single dose)nine-hole peg-test, writing tasks, patient self assessment12 (75%)not reportedshort-lasting foot dystonia (1)Gbadamosi et al. 2001 [39]14open labelondansetron (iv.) 8 mg (single dose)nine-hole peg-test, writing tasks, patient self assessmentno effectnone reportedAisen et al. 1991 [2]6open labelGluthetimide 750 to 1250 mgblinded functional assessment by occupational therapist, computer-aided tracking tasks5 (83%)5 (83%)sedation (4)Clifford, 1983 [21]8open labelΔ9-THC (po.) 5 to 15 mgclinical examination5 (63%)2 (25%)noneZajicek et al. 2003 [103]365†double-blind randomized placebo controlledcannabis extract (po.) Δ9-THC (po.)patient self assessmentno effectcannabis extract: adverse events in 12 Δ9-THC: adverse events in18 placebo: adverse events in 20Wade et al. 2004 [92]13‡double-blind randomized placebo controlledcannabis extract (po.)visual analogue scale symptom reductionno effectcannabis extract:$ 112 adverse events in 80 patients placebo:$ 53 adverse events in 80 patientsFox et al. 2004 [37]14double-blind randomized placebo controlled crossovercannabis extract (po.)tremor rating scaleno effectcannabis extract: adverse events in 10 placebo: adverse events in 2THC = tetrahydrocannabinol* MS patients completing the study** in this study, one additional patient was withdrawn because of a severe adverse event (dyspnoea, fever, rash, obtundation)† the main topic of the study is the effect of cannabinoids on spasticity in MS, the patients were asked to assess treatment effect on other symptoms‡ in this study, patients were asked to name their most troublesome symptom; of the 160 included patients, 13 named tremor$ No details are reported on the subgroup of the 13 tremulous patients, the total number of adverse events is reported for the whole groups on active treatment and on placebo (80 patients in each group)
Beneficial effects have been reported for a variety of drugs: case reports and small uncontrolled open label studies claim a positive effect of primidone [47], gluthetimide [2], intrathecal baclofen [96] and isoniazid. [80, 32, 67, 38] Controlled clinical trials have been published on the use of propranolol [52], ethanol [52], isoniazid, [52, 44, 16] carbamazepine [86], ondansetron [78] and dolasetron [65].
Koller evaluated the effect of propranolol, isoniazid and ethanol on tremor in three tremulous MS patients in a double blind crossover trial and found no treatment effect for any of the treatments [52]. To the best of our knowledge, no further trials with propranolol have been published, but it is interesting to note that Alusi and coworkers excluded two tremulous MS patients from a thalamotomy trial because they had achieved functional improvement after propranolol use [4].
In an open study the hypnotic-sedative drug glutethimide exhibited visible functional benefit in six of eight MS patients with tremor [2]. Apparently, a controlled trial to confirm these findings was never undertaken.
Two double-blind placebo-controlled trials using isoniazid were published. In the first study reduction of tremor occurred in six of eight patients [16], with functional improvement in four patients. In the second study all six patients had measurable tremor reduction after treatment with isoniazid but this did not lead to functional improvement [44]. Doses of isoniazid used to treat MS-related tremor were very high (up to 1200 mg a day), and treatment was in some patients associated with anorexia and nausea [38] or with a combination of drowsiness, dysphagia and increased bronchial secretion. [32, 67] Other reported side effects were abnormal liver function tests, [80, 32] fatigue [32] and increased weakness [67]. Isoniazid inhibits GABA aminotransferase activity and increases GABA in the central nervous system, but no correlation was found between the degree of GABA elevation in the cerebrospinal fluid and clinical response [16].
Sechi and coworkers published a small single-blind placebo-controlled trial with carbamazepine [86]. They reported improvement of tremor as assessed by clinical examination and accelerometry in all seven included patients, but failed to report whether this translated into functional improvement.
A placebo controlled, double-blind, crossover study, suggested that a single intravenous dose of the 5-HT3 receptor antagonist ondansetron led to tremor reduction in twelve, and to functional improvement in nine of the sixteen included tremulous MS patients [78]. A subsequent open label study by Gbadamosi and colleagues which used the same intervention found no significant treatment effect [39]. Likewise, a small clinical trial with dolasetron, another 5-HT3 receptor antagonist, showed no significant treatment effect on cerebellar ataxia [65].
Weiss and coworkers reported a positive effect of intrathecal baclofen in a single patient with bilateral arm tremor [96]. The tremor amplitude decreased almost linearly as the dosage increased and tremor was abolished at a dosage of 250 µg per day. This possible treatment option has, however, not been observed or evaluated in other studies.
In recent years, there has been growing interest in cannabis as a possible therapeutic in MS. In the 1980’s a case report [63] and a small uncontrolled study [21] on the beneficial effects of cannabis on tremor and spasticity in MS were published and a survey taken among MS patients revealed that many patients experienced positive effects of smoked cannabis on MS-related symptoms [23].
Baker and colleagues reported a decrease of tremor and spasticity in an animal model of MS after treatment with Δ9-tetrahydrocannabinol, the active ingredient of cannabis [12]. All this furthered hopes of cannabis as a possible new treatment option for tremor in MS, but much to the disappointment of tremulous MS patients, several well conducted randomized controlled trials did not show a significant effect of orally administered cannabis extracts [103, 37, 92] or oral Δ9-tetrahydrocannabinol [103] on tremor.
Surgical treatment
The surgical treatment options for tremor in MS are stereotactic thalamotomy and DBS. An overview of the published studies on surgical treatment is given in Tables 3 and 4. Most of the studies are small observational retrospective studies. When reviewing the literature on surgical treatment, it is surprising as well as disappointing that the majority of studies are remarkably imprecise in providing basic information on the length of follow-up, on adverse effects and-most importantly-on the effect on functional status and tremor associated disability.
Table 3Studies on stereotactic surgery for the treatment of tremor in MSStudyn*patient characteristics**lesion sitefollow-uptremor and disability assessmentpatients with tremor reduction (%)§patients with improved functional status (%)§patients with permanent adverse effects (n)Cooper, 1960a [25]2disabling intention tremorVL3 to 12 moclinical examination2 (100%)not reportednoneCooper, 1960b [24]6disabling intention tremorunilateral (n = 5) or bilateral (n = 1) VLNot specifically reportedclinical examination, assessment of filmed tremor5 (83%)not reportedincrease of contralateral hemiparesis (1)Krayenbühl et al. 1962 [54]4disabling bilateral intention tremorunilateral (n = 3) or bilateral (n = 1) VL3 weeks to 6 moclinical examination4 (100%)4 (100%)noneBroager and Fog, 1962 [18]4severe intention tremorunilateral VL1 to 6 moclinical examination4 (100%)2 (50%)generalized seizure (1) mental change (1)Cooper et al. 1967 [26]32disabling bilateral intention tremorunilateral or bilateral VL12 to 96 moclinical examination27 (85%)not reportedincrease of contralateral hemiparesis (2)Samra et al. 1970 [81]25disabling bilateral arm intention tremorunilateral or bilateral VLnot specifically reportedclinical examination, assessment of filmed tremor22 (88%)not reportedincrease of contralateral hemiparesis (1)Riechert and Richter, 1972 [79]29disabling intention (n = 29) and postural (n = 18) tremorunilateral (n = 28) or bilateral (n = 1) VLnot specifically reportedclinical examination, patient self assessment questionnaires29 (100%)“two thirds” of patientsincrease of leg paresis (2)Arsalo et al. 1973 [10]26disabling bilateral intention tremorunilateral VL and subthalamus3 to 97 moclinical examination21 (80%)not reportedsubdural haematoma (1)Andrew et al. 1974 [8]4disabling postural (n = 2) and intention (n = 4) tremorunilateral VIM6 to 36 moclinical examination4 (100%)not reportedhemiplegia (1)Van Manen, 1974 [91]4severe intention tremorunilateral VL3 to 86 mo†clinical examination2 (50%)not reportednot reported separately for MS subgroupHauptvogel et al. 1975 [45]11severe intention tremorunilateral (n = 10) or bilateral (n = 1) VL15 to 86 moclinical examination7 (63%)4 (36%)not reportedMundinger and Kuhn 1982 [68]84severe action tremorZI, VOP36 to 120 moclinical examination filmed tremor patient self assessment questionnaire70 (83%)not reportednot specifically reportedSpeelman and van Manen, 1984 [89]11severe intention tremorunilateral VL3 weeks to 132 moclinical tremor and functional rating scales8 (73%)0 (0%)hemiparesis (4) micturition disturbance (2) speech disorder (1)Kandel and Hondcarian, 1985 [51]20severe intention tremorunilateral (n = 15) or bilateral (n = 5) VL (ZI, FF)12 to 120 monot reported14 (70%)not reportednoneHitchcock et al. 1987 [48]30tremorThalamus24 moclinical examination functional rating scales50%25%not reportedWester et al. 1990 [97]9severe intention tremorunilateral VOA and VOP3 to 89 mo mean: 24 mo†questionnaire sent to treating neurologist6 (66%)6 (66%)hemiparesis (5) mental changes (3) dysphasia (3) dysarthria (2) subdural haematoma (1)Goldman et al. 1992 [43]2severe intention tremor unresponsive to medicationunilateral VL3 mo 34 moclinical tremor rating scale2 (100%)0 (0%)dysarthria (1)Whittle and Haddow, 1995 [98]9severe rest, kinetic, postural or intention tremorunilateral VL12 moclinical examination and evauation of video tapes9 (100%)2 (22%)depression (2)Shahzadi et al. 1995 [87]33severe tremorunilateral VIM3 to 120 moclinical examination ability to drink from a waterfilled cup22 (67%)17 (51%)not reportedHooper and Whittle, 1998 [50]6severe postural (n = 4) or intention tremor (n = 2)unilateral VL14 to 73 mo mean: 51 moBarthel Index1 (16%)1 (16%)not reportedCritchley and Richardson, 1998 [27]24disabling intention tremorunilateral (n = 22) or bilateral (n = 2) VIMmean: 26 moclinical tremor and functional rating scales18 (75%)2 (8%)hemiparesis (1) seizure (2) MS relapse (3) dysarthria (1)Schuurman et al. 2000 [85]5severe arm tremorunilateral VIM‡6 moclinical tremor and functional rating scales5 (100%)0 (0%)severe gait or balance disturbance (2)Niranjan et al. 2000 [72]***3severe action tremorunilateral VIM2 to 11 mo median 6 mo†clinical tremor rating scale, patient self assessment of functional improvement3 (100%)3 (100%)noneAlusi et al. 2001 [4]11severe postural and intention tremorunilateral VOP (n = 7), ZI (n = 3), STN (n = 1)12 moclinical tremor and functional rating scales11 (100%)7 (64%)depression (3) seizures (2) hemiparesis (1) dysphasia (1)Matsumoto et al. 2001 [62]6severe tremorunilateral VIM3 to 12 moclinical tremor and functional rating scales, novel movement analysis tool6 (100%)0 (0%)MS relapse (2)Bittar et al. 2005 [15]10disabling postural and intention arm tremorunilateral VOP (distal tremor), unilateral ZI (proximal tremor) or unilateral VOP and ZI (mixed tremor)12 to 50 mo: mean 16 moclinical tremor rating scalenot individually reported, overall improvement of mean tremor scores: postural: 78% intention: 72%not reportedhemiparesis (3) seizures (1)VOP = nucleus ventralis oralis posterior; VOA = nucleus ventralis oralis anterior; VIM = nucleus ventralis intermedius; ZI = zona incerta; VL = nucleus ventralis lateralis; STN = nucleus subthalamicus; FF = Forel’s Field* MS patients with completed surgical intervention and remaining in the study until end of follow up** an effort is made to distinguish between predominance of intention or postural tremor although many terms to describe tremor subtypes are used in the studies*** In this study, gamma-knife radiosurgery is used§ improvement as described in case reports or measured at the end of follow-up on any scale used in the study† in this study, MS patients were grouped together with patients with other movement disorders, no details are given for the MS-subgroup‡ thalamotomy was followed six months later by contralateral DBS electrode implantation in patients with bilateral tremorTable 4Studies on DBS for the treatment of tremor in MSStudyN*patient characteristicsstimulation sitefollow-uptremor and disability assessmentpatients with tremor reduction (%)§patients with improved functional status (%)§patients with permanent adverse effects (n)Brice and McLellan, 1980 [17]2severe bilateral arm intention tremorbilateral subthalamic5 mo 6 moclinical examination2 (100%)2 (100%)**Nguyen and Degos, 1993 [71]1severe unilateral distal postural arm tremorunilateral VIM17 moclinical tremor and functional rating scales1 (100%)1 (100%)not reportedSiegfried and Lippitz, 1994 [88]9severe intention tremorunilateral (n = 8) or bilateral (n = 1) VIMnot reportednot reported9 (100%)not reportednot reportedBenabid et al. 1996 [13]4severe arm tremor†VIM†≥ 6 moclinical tremor rating scale0 (0%)‡no detailed report†intracerebral haemorrhage (1)Geny et al. 1996 [41]13severe postural tremor (n = 12), moderate intention tremor (n = 1)unilateral VIM8 to 26 mo mean: 13 moclinical tremor and functional rating scales9 (69%)12 (92%)MS relapse (3)Whittle et al. 1998 [99]***5severe arm tremorVLnot reportednot reportednot reportednot reportednot reportedHay, 1999 [46]1head and limb tremorunilateral thalamus2 monot reported1 (100%)not reportednot reportedMontgomery et al, 1999 [66]14disabling arm tremorunilateral VIMvariableclinical tremor rating scale15 (100%)not reportedMS relapse (1)#Schulder et al. 1999 [83]5severe bilateral postural and intention arm tremorunilateral VIM>6 moclinical tremor rating scale, patient self assessment of functional improvement5 (100%)3 (60%)Ms relapse (2)Taha et al. 1999 [90]2bilateral limb, head or voice tremor†bilateral VIM (bilateral DBS or unilateral DBS plus contralateral thalamotomy)mean: 10 moclinical tremor rating scale2 (100%)not reportednot reported separately for MS subgroup†Schuurman et al. 2000 [85]5severe arm tremorunilateral or bilateral VIM6 moclinical tremor and functional rating scales3–5 (60–100%)$0 (0%)dysarthria (2) severe gait or balance disturbance (1) arm ataxia (1)Krauss et al. 2001 [53] Loher et al. 2003 [61]$$2severe tremorunilateral or bilateral VIM†3 to 24 mo, mean: 12 mo†clinical tremor rating scales, assessment of video tapes2 (100%)not reportednot reported separately for MS subgroup†Matsumoto et al. 2001 [62]3severe tremorunilateral VIM3 to 12 moclinical tremor and functional rating scales, novel movement analysis tool3 (100%)0 (0%)noneHooper et al. 2002 [49]10disabling arm tremorunilateral thalamus12 moclinical tremor and functional rating scales10 (100%)0 (0%)intracerebral haemorrhage (2) generalized seizure (2)Nandi et al. 2002 [70]1severe bilateral postural and intention tremorunilateral ZI12 moclinical examination1 (100%)1 (100%)increased dystonic posturing of left foot impairing ambulation (1)Berk et al. 2002 [14]12disabling arm tremorunilateral VIM12 moclinical tremor and functional rating scales, patient self assessment questionnairesignificant tremor reduction, not individually reportedno significant improvementwound infection (2)Wishart et al. 2003 [102]4bilateral arm tremorbilateral VL15 to 31 moclinical tremor rating scale4 (100%)4 (100%)MS relapse (1) dysarthria (1)Schulder et al. 2003 [84]9disabling arm tremorunilateral thalamus9 to 48 mo,clinical tremor and functional rating scales, patient self assessment8 (88%)3 (33%)MS relapse (3)Nandi et al. 2004 [69]10disabling arm tremorunilateral (n = 6) or bilateral (n = 4) VOP and ZI3 to 23 mocomputer-aided tracking taskssignificant tremor reduction, not individually reportednot reportedseizure (1) dysarthria (1) wound infection (1)Bittar et al. 2005 [15]y10disabling postural and intention arm tremorunilateral VOP (distal tremor), unilateral ZI (proximal tremor) or unilateral VOP and ZI (mixed tremor)3 to 23 mo:clinical tremor rating scalenot individually reported, overall improvement of mean tremor scores: postural: 64% intention: 36%not reportedmonoparesis (1)VIM = nucleus ventralis intermedius; ZI = zona incerta; VL = nucleus ventralis lateralis* MS patients with completed surgical intervention and remaining in the study until the end of follow up§ improvement as described in case reports or measured at the end of follow-up on any scale used in the study** in this study two patients had complete surgery and in three, surgery was aborted. A worsening of dysarthria is reported in one patient, it is unclear whether this patients had completed surgery*** This study mostly discusses difficulties in target localisation and patient selection# in this study, one patient did not complete surgery due to an intraoperative VIM haemorrhage† in this study, MS patients were grouped together with patients with other movement disorders, no details are given for the MS-subgroup‡ For the whole group, tremor was “inconsistently, less significantly [than tremor of Parkinson’s Disease and essential tremor] or not improved. If improvement was achieved it lasted only a few months”$ The exact number of patients with improved tremor cannot be ascertained the way the data is presented in this study$$ Both studies are on one patient cohort, the relevant data on MS patients are derived from both studies
The first study on thalamotomy for tremor in MS was published by Cooper in 1960 [25]. Brice and colleagues were the first to report improvement of tremor through continuous thalamic DBS in 1980 [17].
Strategies for patient and treatment site selection
The earlier studies on thalamotomy used the thalamic nucleus ventralis lateralis (VL) as the target, whereas the nucleus VIM was chosen in most DBS studies and most later thalamotomy studies. This preference for the VIM is probably due to the experience with this thalamic nucleus in the treatment of tremor in Parkinson’s disease (PD) and essential tremor (ET). Research groups in Oxford and London used the nucleus VOP and the zona incerta (ZI) as targets for both lesional surgery and DBS, but the results yielded with these targets [4, 69, 15] were not different from those reported for VIM or VL thalamotomy or stimulation.
The same researchers advocate the use of tremor frequency analysis during movement tasks as a method to identify patients likely to benefit from surgery [60]. This may be a valuable tool for patient selection, although it has only been validated in a small number of patients and studies in which this technique is used [4, 69, 15] do not report better results than studies without this selection method.
The site for lesional surgery or electrode placement is classically chosen relative to the site of the anterior and posterior commissures using a standardized atlas. More recently, surgeons tried to refine this placement strategy with microelectrode recording within the target area. Neurons discharging synchronous to peripheral tremor are identified and the treatment site is placed in an area where the most tremor related neuronal activity is found. [73, 59, 57, 58]
This method of treatment site selection is often used in studies on tremulous patients with PD and ET, but only a small number of MS-patients were included in these studies. [87, 13, 66, 62, 84] Since this method offers a theoretical advantage over the classical methods of treatment site selection, it should be further evaluated.
Outcome after surgical treatment
Because of the many shortcomings of the published studies, the results need to be interpreted with great caution. It does, however, seem as if almost all patients experience tremor reduction immediately after thalamotomy, and roughly 70% of patients continue to benefit from thalamotomy beyond a follow-up period of one year (Table 3). In the three studies on thalamic DBS with a follow-up period longer than one year, 69% to 100% of the patients experienced reduced tremor [41, 84, 102]. Functional improvement after both thalamotomy and DBS is much more variable and unfortunately not reported in many studies (Tables 3 and 4).
Niranjan published an interesting study on gamma-knife thalamotomy as a possible alternative to neurosurgery. In this study all three patients with MS-related tremor experienced marked improvement of tremor after radiosurgery. Unfortunately, no further studies have been published to evaluate this interesting non-invasive treatment option [72].
There are two trials in which thalamotomy and DBS were compared in patients with MS. In a randomized controlled trial conducted by Schuurman and colleagues [85] patients with ET, tremor due to PD and tremulous MS patients were randomized to undergo either VIM thalamotomy or DBS. Five tremulous MS patients were randomized to each group. After a short follow-up period of six months, there were no significant differences between thalamotomy and DBS in functional outcome for the MS subgroup. In the same study, fewer adverse effects were observed in the DBS group, except for the subgroup of MS patients, where adverse effects were about equal for both interventions (Tables 3 and 4). Bittar and colleagues compared cumulative tremor scores for intention and postural tremor between patients undergoing VOP/ZI thalamotomy or DBS in a non-randomized study [15]. Ten patients were in each group. Interestingly, after a mean follow-up period of 15 to 16 months, patients in the thalamotomy group had a better outcome (tremor reduction: 78% for postural tremor and 72% for intention tremor) than patients in the DBS group (tremor reduction: 64% for postural tremor and 36% for intention tremor). However, as there were more adverse effects in the thalamotomy group, no clear recommendations could be given.
Adverse events reported for the neurosurgical interventions comprised increase of hemiparesis, dysarthria, dysphasia, mental changes, depression, seizures, intracerebral haemorrhage, subdural haematoma, wound infection and MS relapse. Thalamotomy was associated with a higher risk of adverse events than DBS. Bilateral thalamotomy carries such a high risk of adverse effects, that it is no longer recommended. If bilateral treatment is necessary, either bilateral DBS or unilateral thalamotomy followed by contralateral DBS are possible treatment options.
Other treatment options
Electromagnetic fields, limb cooling, physiotherapy, weight bracelets, orthoses and specialized software have been advocated as additional treatment options.
Sandyk and Dann reported a reduction of intention as well as postural tremor in three tremulous MS patients treated with pulsed electromagnetic fields [82], but these interesting findings have not been substantiated in a larger trial.
Albrecht and coworkers published a small clinical trial on the effect of arm cooling on intention tremor [3]. In their study, patients achieved significantly better results on a clinical testing battery after immersion of the tremulous arm in ice water. As this effect lasted for about 45 minutes the authors recommend limb cooling to achieve transient tremor control for activities such as working with a PC, signing a document or self-catherisation. In a similar study, Feys and colleagues report tremor reduction lasting for about 30 minutes after limb cooling with a special cooling device [35].
Weighted wrist cuffs are a mechanical tool to reduce tremor amplitude, and one article reported their beneficial effects in three MS patients [28]. Although wearing bracelets decrease tremor amplitude and therefore may offer some benefit to tremulous patients, their effect on intention tremor is small and their use is therefore unlikely to yield important functional improvement. A more advanced computer aided tremor reducing orthosis provided functional benefit in a small case series including patients with MS [1].
Physiotherapy aimed at improving ataxia in MS was evaluated by Armutlu and coworkers. In their small pilot study they found that rehabilitative physiotherapy using Johnstone pressure splints was superior to physiotherapy alone [9]. Unfortunately no larger trials followed this pilot study, so that it remains uncertain which patients may benefit from which form of physiotherapy.
The use of a mouse driven computer system is a special challenge for tremulous MS patients. Feys and colleagues published a study on the use of specialized software developed to aid computer use in 36 tremulous MS patients and found significant improvement in the time needed to complete some basic mouse driven computer operations [36].
Conclusion
The treatment of tremor remains a great challenge for everyone caring for patients with MS. Drug treatment with currently available medication is unsuccessful in most cases and much more research on the pathophysiology and biochemistry of tremor production in MS will be necessary before an efficient medical treatment can be developed. Stereotactic surgery can be an effective means to treat severe tremor, but it is currently uncertain whether lesional surgery or DBS is the treatment of choice. Larger clinical trials comparing both interventions are needed. Other treatment options, including physiotherapy, tremor reducing orthoses, and limb cooling can lead to valuable improvements in activities of daily living.

tremor
multiple sclerosis
ataxia
treatment


Reperfusion injury following cerebral ischemia: pathophysiology, MR imaging, and potential therapies

Introduction Restoration of blood flow following ischemic stroke can be achieved by means of thrombolysis or mechanical recanalization. However, for some patients, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called “cerebral reperfusion injury”. Multiple pathological processes are involved in this injury, including leukocyte infiltration, platelet and complement activation, postischemic hyperperfusion, and breakdown of the blood–brain barrier.
Introduction
In the treatment of acute stroke, restoration of the blood supply can reduce more extensive brain tissue injured by salvaging a reversibly damage penumbra of tissue [1]. This mechanism provides a rationale for clinical trials which have demonstrated that reperfusion after thrombolysis improves clinical outcome in selected patients with acute stroke [2].
Reperfusion, however, carries certain risks. Some patients experience disastrous outcomes in the form of fatal edema or intracranial hemorrhage following thrombolysis [3]. In some animal stroke models [4, 5], reperfusion after a long ischemic period can cause a larger infarct than that associated with permanent vessel occlusion. Thus, while reperfusion may reduce infarct size and improve clinical outcome in some patients, in others it may exacerbate the brain injury and produce a so-called “cerebral reperfusion injury” [4, 6, 7]. Cerebral reperfusion injury can be defined as a deterioration of ischemic but salvageable brain tissue after reperfusion.
Thrombolysis [2] and embolectomy [8, 9] usually result in reperfusion of the infarcted brain tissue and therefore carry the risk of causing reperfusion injury. Thus reperfusion injury deserves the attention of those interested in the diagnosis and treatment of acute stroke. Strategies to reduce or minimize cerebral reperfusion injury require the understanding of the pathophysiology of cerebral reperfusion injury, and the way the reperfusion injury is visualized by magnetic resonance imaging (MRI). Therapeutic options for preventing or attenuating cerebral reperfusion need to be considered.
Cerebral reperfusion injury: mechanisms and correlation with MRI
Leukocyte infiltration and secondary ischemia as depicted with DW MRI
Leukocytes play an important role in the development of cerebral reperfusion injury. During reperfusion, activated leukocytes interact with endothelial cells and plug capillaries, disrupt the blood-brain barrier (BBB) through the release of neutrophil-derived oxidants and proteolytic enzymes, extravasate from capillaries and infiltrate brain tissue, and release cytokines which mediate inflammation. These processes produce an inflammatory cascade, resulting in the deterioration of the salvageable penumbra [7].
Evidence of the deleterious effects caused by leukocytes is provided by animal studies. For example, Zhang et al. [10], using a rat model of 2 hours transient focal cerebral ischemia induced by advancing a nylon monofilament to occlude the middle cerebral artery (MCA), showed that neutrophils accumulate at the site of neuronal injury 6 hours after restoration of cerebral circulation. The neutrophil accumulation occurred earlier and to a greater extent in reperfusion tissue than in tissue permanently deprived of blood supply [10]. Furthermore, this study showed that the infarct volume increased dramatically between 6 and 24 hours following the start of reperfusion, and that the period of maximal infarct expansion correlated closely with the time course of neutrophil infiltration.
The contribution of leukocytes to cerebral reperfusion injury is also supported by the beneficial effects of neutrophil depletion. Bednar et al. [11] investigated the effect of administering antineutrophil antiserum treatment on brain infarct size in a rabbit model of transient ischemia. The regional cerebral blood flow (CBF) of neutropenic rabbits recovered from less than 5 ml/100 g per minute to 20–30 ml/100 g per minute following reperfusion, while in the non-neutropenic rabbits it remained at less than 10 ml/100 g per minute. Correspondingly, the infarct size was significantly smaller in the neutropenic animals. Similar results have been reported in rats treated with antineutrophil monoclonal antibodies [12].
The evidence from animal experiments points to the role of neutrophils in restricting CBF and increasing infarct size during reperfusion. High-quality evidence of reperfusion-induced secondary ischemic injury has been obtained using diffusion-weighted imaging (DWI) in animals. Furthermore, the evolution of reperfusion injury can be depicted by observing dynamic changes with DWI (Fig. 1) [13].
Fig. 1DWI images of a rat model with 1 hour of MCA occlusion (MCAO) followed by 10 hours of reperfusion. These images show the evolution of ADC at different times: before ischemia (control), at the end of MCAO, and at different time points of reperfusion. Note the transient recovery of ADC during the early phase of reperfusion, followed by secondary deterioration (reproduced with permission from Olah et al. [13])
Olah et al. [13] investigated the dynamics of the apparent diffusion coefficient (ADC) in a rat model with 1 hour of MCA occlusion (MCAO) followed by 10 hours of reperfusion. They found that the ADC was decreased at the end of ischemia, then significantly improved during the first 2 hours of reperfusion (Fig. 2), but decreased again at later time points with a secondary increase of hemisphere lesion volume. The ADC change most likely reflects a secondary ischemic injury. Although a potential role for apoptosis cannot be excluded, the observed secondary deterioration of ADC did not support apoptosis as a major mechanism, because the ADC decline indicated cell swelling and not loss of cell volume, which is a fundamental and universal characteristic of programmed cell death [14]. Similarly, Neumann-Haefelin et al., also using a rat MCAO model, showed that DWI abnormalities reversed transiently during the early reperfusion period but recurred after several hours of restored blood flow [15]. Likewise, Li et al., using a rat model with 10–30 minutes of temporary MCAO, observed that the DWI hyperintensity seen during the initial ischemic insult reverted to normal approximately 60–90 minutes after the start of reperfusion, with secondary hyperintensities emerging 12 hours after reperfusion [16]. The secondary increase of DWI signal intensity observed in these studies is likely a marker of cerebral reperfusion injury [15].
Fig. 2Temporal evolution of mean relative ADC in the end-ischemic lesion volume in a rat model with 1 hour of MCA occlusion (MCAO) followed by 10 hours of reperfusion. Although a significant improvement in ADC attends reperfusion, there is a secondary deterioration likely as a consequence of the reperfusion-related injury (reproduced with permission from Olah et al. [13])
Several interesting characteristics of the ADC changes during reperfusion have been reported in animal studies. First, the time of onset of the secondary reduction of ADC varies amongst different animal studies, ranging from 2–72 hours after reperfusion [12, 16–18]. The reason is probably due to the different durations of the initial ischemic insult and differences in the animal model used. Second, the secondary deterioration of ADC can be predicted by early changes of T2 during reperfusion. Olah et al. [13], in their rat model study, observed that an increase in T2 value early in the ischemic period predicts a secondary deterioration in ADC after reperfusion. This suggests the possibility of using T2 value clinically to predict the extent of reperfusion injury likely to follow recanalization, and may be useful in assessing patients’ likelihood of benefiting from thrombolysis.
Hemorrhagic transformation (HT) follows hyperperfusion and disruption of the BBB (see below), but some of the imaging studies are reviewed in this section because there is a close relationship between the ADC changes and the subsequent HT after cerebral ischemia. In a rabbit model of thromboembolic stroke, Adami et al. [19] compared the percentage of pixels with very low ADC at various time points after the start of ischemia with the subsequent occurrence of HT (as determined on histopathological sections). The authors found that, at each time point, regions populated by pixels with an ADC value below the threshold of 550 × 10−6 mm2/s were likely to undergo HT, and that the percentage of pixels below this threshold correlated with the severity of HT. This result has also been confirmed in several human studies. In a group of acute stroke patients treated with intravenous recombinant tissue plasminogen activator (rtPA) within 3 hours from symptom onset, Selim et al. [20] demonstrated that the absolute number of voxels with ADC values ≤550 × 10−6 mm2/s was much higher in patients with HT than in those without. The authors suggested that, although patients with both small and large DWI lesions have the tendency to develop HT, the volume of ischemic tissue with ADC ≤550 × 10−6 mm2/s may best predict the risk of intracerebral HT after thrombolysis.
ADC values have also been reported to correlate with recanalization and clinical outcome in ischemic stroke patients undergoing local intra-arterial thrombolysis. Taleb et al. [21] identified two patient groups on angiographic findings: one group with more than 50% recanalization and the other with less than 50%. Relative ADC values were found to be higher in the first group of patients. Moreover, all patients in the recanalization group had a Barthel index of 80 or more, whereas eight of ten patients in the nonrecanalization group had a Barthel index of 50 or less.
Molecular mechanisms of leukocyte infiltration
The mechanisms of leukocyte infiltration are characterized by leukocyte activation, leukocyte–endothelial interaction resulting in accumulation in the vascular bed, followed by leukocyte extravasation into the interstitial space. Each of these mechanisms is briefly reviewed.
The accumulation of leukocytes, along with red blood cells and platelets during reperfusion, may plug capillaries, preventing the restoration of blood flow and resulting in a “no-reflow” phenomenon and secondary cerebral ischemia [22–27]. Del Zoppo et al. [27] used a microscopic imaging system in a baboon model of transient ischemia. There was significant capillary obstruction caused by leukocytes reperfusing the microvascular bed within 60 minutes after the restoration of cerebral circulation. The “no-reflow” phenomenon explains well the better recovery of regional CBF in neutropenic rabbits [11].
The molecular mechanisms by which leukocytes infiltrate the parenchyma during reperfusion have been elucidated. This multistage process can be conceptually divided as follows: (1) leukocytes “rolling” on the endothelium, (2) firm adherence of leukocytes to the endothelium, and (3) transmigration into the tissue [1, 28–31]. Superoxide free radicals produced during ischemia and reperfusion cause the upregulation of endothelial P-selectin. Once leukocytes reach the ischemic territory the endothelial P-selectin interacts with its leukocyte counter-receptor P-selectin glycoprotein 1 [31]. This initial interaction facilitates the low affinity “rolling” of leukocytes on the endothelium. Firm adherence is developed with subsequent interaction of the leukocyte β2 integrins CD11a/CD18 and CD11b/CD18 with endothelial intercellular adhesion molecule 1 (ICAM-1).
The transmigration of leukocytes results from the expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) along the endothelial cell junction. Once the leukocytes infiltrate the parenchyma, they then release various chemical mediators including neutrophil elastase, reactive oxygen species (ROS), leukotrienes, and prostaglandins, resulting in increased microvascular permeability, edema, thrombosis and parenchymal cell death [1, 27, 32]. This process compounds the initial injury that occurred in these tissues during the hypoxic insult.
Platelet-mediated reperfusion injury
Platelets play a synergistic role with leukocytes in reperfusion injury. Several studies suggest that platelets are activated after cerebral ischemia and reperfusion [33–35]. Following activation, platelets may adhere to both leukocytes and cerebral microvascular endothelial cells, contributing to the “no-reflow” phenomenon and furthering tissue injury. Additionally, activated platelets release a variety of biochemical mediators of tissue injury.
Several studies have confirmed that platelet activation occurs in patients with stroke. Zeller et al. [36] used flow cytometric detection of platelet adhesion molecules as a marker of platelet activation, and reported a substantially greater activation in acute stroke patients than in normal controls. Similarly, van Kooten et al. [37], using urinary excretion of 11-dehydro-thromboxane-B2 as a marker of platelet activation, demonstrated a 347% increase in the marker excretion in ischemic stroke patients compared with matched controls.
Chong et al. [38] investigated changes in platelet–leukocyte adhesion using a rat model of transient MCA ischemia. The authors reported that platelet–neutrophil adhesion was significantly increased from 16.6% in the control group to 39% in the reperfusion group (P < 0.01). Platelets in the stroke group expressed high levels of P-selectin. It was hypothesized that enhancement of platelet–leukocyte adhesion may be mediated by increased P-selectin expression on the platelet membrane.
Studies on the role of platelets in coronary reperfusion of the heart suggest that, in addition to causing mechanical obstruction, platelets can cause temporary vasospasm by releasing serotonin, thromboxane A2, and free radicals. This can cause diminished reperfusion of the ischemic territory [33]. Further, activated platelets can release mediators that may cause chemotaxis and migration of leukocytes, thus exacerbating the inflammatory cascade [33]. These mechanisms may also prove to be important in cerebral reperfusion injury after ischemic stroke.
Complement-mediated reperfusion injury
Experimental studies have shown that complement activation is an important component of reperfusion injury in several organ systems. Complement may be activated during reperfusion through the antibody-dependent classical pathway, the alternative pathway, or the MBL/MASP (mannan-binding lectin/mannan-binding lectin-associated serine proteases) pathway [39, 40]. Complement activation results in the formation of several inflammatory mediators, including anaphylatoxins C3a, C5a, and the distal complement component C5b-9 (membrane attack complex, MAC) [39]. C5a and the MAC are the best-characterized effectors of tissue damage within the complement system [41, 42]. C5a may stimulate leukocyte infiltration into the ischemia and reperfusion injury-affected tissue by its strong chemotaxis action. C5a may also further amplify the inflammatory response by inducing the release of several proinflammatory cytokines, including IL-1, IL-6, monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor α (TNF-α). MAC can cause cellular membrane injury directly and can increase cell membrane permeability. Furthermore, MAC plays an essential role in mediating the recruitment of leukocytes to the reperfused tissue via local induction of IL-8. Several agents that inhibit all or part of the complement components such as cobra venom factor (CVF) [43], C5a monoclonal antibodies [44], and Cl esterase inhibitor [45], have been shown to significantly attenuate the degree of reperfusion injury in animal models, providing further evidence that complement plays an important role in reperfusion injury.
Postischemic hyperperfusion and PWI
Postischemic hyperperfusion is recognized as a frequent occurrence in stroke. Several animal studies suggest that the restoration of cerebral circulation consistently results in a hyperperfusion phase. Hyperperfusion may contribute to the development of reperfusion injury by causing brain edema or hemorrhage. Further, following postischemic hyperperfusion, a phase of secondary hypoperfusion can occur, which results in harmful effects on the reperfused tissue [1, 46, 47].
Heiss et al. [46] observed the effects of hyperperfusion in cats using a model of temporary MCA occlusion of varying durations (30, 60, and 120 minutes). In the 30-minute group, only a transient reactive hyperperfusion (compared with preocclusion values) was found. All animals in this group survived during the observation period, and no cerebral injury was found on histological study. In the 60- and 120-minute groups, the degree of hyperperfusion was significant, reaching up to 300% of preocclusion values. Most importantly, a clear difference was found between surviving and dying cats. In dying cats, postischemic hyperperfusion was long and severe, large infarcts developed and intracranial pressure increased fatally. In surviving cats postischemic hyperperfusion was transient and less severe, infarcts were smaller and intracranial pressure was significantly lower. A report of a similar study design suggested that the degree of hyperperfusion correlates significantly with occurrence of intracerebral petechial hemorrhages in the damaged cortex [47].
Perfusion-weighted imaging (PWI) provides quantitative and qualitative maps of CBF, cerebral blood volume (CBV), and mean transit time (MTT), allowing identification of regions of relative hypo- and hyperperfusion [48]. Kidwell et al. [48] used PW MRI to characterize hyperperfusion in 12 patients following intra-arterial thrombolysis. Hyperperfusion was visualized in 5 of 12 patients. On day 7, 79% of voxels with hyperperfusion demonstrated infarction, whereas only 36% not showing hyperperfusion demonstrated infarction. Despite the voxel-by-voxel association of hyperperfusion with infarction, there were no significant differences in the degree of clinical improvement in patients with regions of hyperperfusion versus those without. It has been reported that reduction in volume of hypoperfused brain on PWI is a marker of response to treatment and improved function in ischemic stroke patients [49]. Larger future studies are necessary to assess the extent to which both hyperperfusion and hypoperfusion may result in unfavorable clinical outcome.
Breakdown of the BBB and contrast-enhanced MRI
Breakdown of the BBB during cerebral reperfusion may lead to the development of vasogenic edema, HT and infarction, all contributing to cerebral reperfusion injury. This hypothesis has been supported by several clinical and animal studies [5, 50–52].
Sage and Duffy [50] investigated the increase in BBB permeability after transient global cerebral ischemia in a rat model. Permeability of the BBB was increased at 15 minutes after reperfusion of the brain, and cerebral edema occurred 15–30 minutes after reperfusion. Another study reported a disruption of the BBB after 3 hours of reperfusion, following a 3-hour temporary occlusion [5]. In contrast, the BBB remained intact after 6 hours of occlusion in a permanent occlusion group. Moreover, the temporary occlusion group had increased ipsilateral hemispheric water content, suggesting that cerebral edema had occurred. In addition to these reports, numerous histological studies with various tracers have established that reperfusion may exacerbate vasogenic edema due to BBB disruption [6, 53, 54].
T2-weighted imaging is useful for detecting vasogenic edema. Neumann-Haefelin et al. [15] used several MRI techniques to monitor the dynamics of secondary ischemic damage, BBB disturbances, and development of vasogenic edema during the reperfusion phase after focal cerebral ischemia in rats. After 2.5 hours of ischemia, signal intensity on both DWI and T2-weighted images increased rapidly in the previously ischemic region due to BBB disruption (enhancement on postcontrast T1-weighted images) and edema formation. More importantly, early BBB damage after reperfusion appeared to be predictive of relatively pronounced increases in T2 lesion size due to vasogenic edema at subacute time points (1 to 2 days).
Using a novel MRI marker that can image BBB disruption, Latour et al. [55] studied a total of 144 acute stroke patients, to test the association between reperfusion, HT and clinical outcome. Nearly two-thirds (63%) of the patients had evidence of reperfusion within 1 week. BBB disruption was more common in patients who reperfused (45%) than in patients who did not reperfuse (18%). Both HT and BBB disruption were more common in patients undergoing intra-arterial rtPA therapy (31% and 55%), than those not treated (14% and 25%). In the reperfused group, patients with BBB disruption were more likely to have a poor clinical outcome (63%) than those without disruption (25%). Moreover, early BBB disruption was an independent predictor of HT. This was a key study because it associated early BBB disruption with HT and poor clinical outcome in humans.
Contrast-enhanced T1-weighted MRI can detect disruption of the BBB during reperfusion. Under normal physiological conditions, contrast agent does not cross the intact BBB. Mechanisms that cause BBB disturbances, such as reperfusion injury, result in leakage of the contrast agent into the brain. In a recent study, rats were subjected either to permanent MCA occlusion, reperfusion after 1 hour (early reperfusion), or reperfusion after 3 hours of MCAO (late reperfusion) [56]. This protocol allowed monitoring the evolution of BBB disruption during permanent or temporary MCAO. Contrast agent extravasation was found in all animals with permanent MCAO and late-reperfusion animals, but in only 40% of early-reperfusion animals. Postischemic hyperperfusion was demonstrated in late-reperfusion animals, but not in early-reperfusion animals. Thus, using contrast-enhanced MRI, the disruption of the BBB was correlated with the hemodynamic and biophysical consequences of reperfusion.
Fenstermacher et al. [57] employed the Patlak plot processing of gadolinium-shifted T1 relaxation-time images to obtain quantitative estimations of BBB opening in rats. Following 2.5 hours of reperfusion, the “blood-tissue distribution” of gadolinium-T1-shifted protons was serially imaged every 3–4 minutes for 20–30 minutes. After that, the value of blood-to-brain transfer constants (K1) for gadolinium was calculated from the serial changes of the relaxation time via Patlak plots [58] of MRI-T1 datasets. The K1 for gadolinium in ischemia and reperfusion areas was around 200 times larger than that of normal areas, indicating a huge impairment of BBB function during ischemia and reperfusion. This approach may be utilized to monitor the time-course of BBB function in transient cerebral ischemia.
The disruption of the BBB has also been observed in ischemic stroke patients undergoing rtPA therapy. A retrospective study of 213 patients used the postcontrast enhancement of the cerebrospinal fluid (CSF) in FLAIR images (termed hyperintense acute reperfusion marker, HARM) to characterize early BBB disruption [59]. BBB disruption was more common in patients with reperfusion (45%) than in patients without reperfusion (18%, P = 0.006). In multiple logistic regression, reperfusion was the strongest independent predictor of early BBB disruption (OR 4.09, 95% CI 1.28 to 13.1, P = 0.018). HARM was associated with HT and worse clinical outcome. This is a key study for two reasons: first, the association between reperfusion, BBB disruption and HT was established in humans, and second, it may be possible to reduce the complications of rtPA by targeting adjunctive therapies against BBB disruption. In another recent study, three of the six patients receiving rtPA showed early parenchymal enhancement, and all three of them went on to develop HT, whereas none of the patients without early parenchymal enhancement developed hemorrhage [60]. The results suggest that early parenchymal enhancement may be significantly correlated with subsequent symptomatic HT and may be helpful in identifying patients at risk after thrombolytic therapy.
Potential therapeutic strategies
With the progress made in the understanding of the mechanisms in cerebral ischemia and reperfusion injury, an increasing number of strategies have been developed for limiting or preventing further brain damage during reperfusion [61–64]. Many of these strategies interfere with leukocyte infiltration. Two previously described animal studies indicated that leukocyte depletion with either antineutrophil antiserum treatment or with antineutrophil monoclonal antibody leads to better recovery of regional CBF and significantly reduces infarct size after cerebral reperfusion [11, 12]. Similarly, anti-ICAM-1 monoclonal antibodies reduce neutrophil infiltration and lesion size after reperfusion in rats, while they are ineffective in permanent ischemia [65], suggesting that ICAM-1 may be an attractive therapeutic target that specifically addresses the problem of reperfusion injury.
However, the translation of these promising antileukocyte strategies into effective therapies in humans has been disappointing. A randomized controlled trial (RCT) evaluated the use of enlimomab, a murine ICAM-1 antibody, in 625 ischemic stroke patients [66]. Patients were randomized to receive either enlimomab for 5 days or placebo within 6 hours after stroke onset. At 90 days, the modified Rankin scale score was worse in patients treated with enlimomab. Moreover, the hazard of death was 43% higher on enlimomab than on placebo. There are several possible explanations for the failure of enlimomab to produce the desired effects. It is possible that the positive results seen with anti-ICAM therapy in animal studies do not reflect the immunological response in human stroke. Alternatively, ICAM-1 therapy may be effective only after reperfusion [65], and it is believed that only a minority of patients experience complete reperfusion after ischemic stroke [67]. Thus, it may have been more appropriate to try enlimomab in patients who received thrombolytic therapy.
Another RCT randomized 966 ischemic stroke patients presenting within 6 hours of acute stroke to either placebo or UK-279,276, a CD11b/CD18 inhibitor. Of these patients, 204 also received tPA when indicated [68]. The trial was prematurely terminated when an interim analysis showed that the trial would be unable to demonstrate the benefit of this therapy in terms of improvement in Scandinavian Stroke Scale score at 90 days.
Some conventional drugs have been shown in animal experiments to provide effective neuroprotection. For example, studies in a rat model have demonstrated that both dextran sulfate and unfractionated heparin can effectively prevent leukocyte infiltration and reduce infarct volume after reperfusion [69, 70]. Although the actual mechanism is still unclear, these findings suggest a possible role for these agents alongside other therapeutic modalities that cause recanalization (i.e. intravenous/intra-arterial rtPA, mechanical thrombolectomy) [69, 70]. Trials investigating this approach in humans have yet to be conducted. Besides these drug therapies, there are two management strategies, brain cooling and conditioned blood perfusion, which are reviewed in some detail because they can modulate several pathways/mechanisms that contribute to reperfusion injury. Both strategies are promising, and interventional neuroradiologists may have a role in administering these therapies in the future.
Brain cooling
Reports on the role of hypothermia in humans are encouraging. Two large RCTs established conclusively the neuroprotective effects and safety of systemic hypothermia after cardiac arrest [71, 72]. A meta-analysis of the results [73] showed that patients in the hypothermia group were more likely to be discharged with no or only minimal neurological damage (risk ratio 1.68; 95% CI 1.29–2.07). This translated to six as the a number needed to treat in order to prevent one death or serious neurological deficit.
There are no hypothermia efficacy studies in ischemic stroke patients. Several animal studies of temporary MCAO have shown that systemic hypothermia applied up to 3 hours after reperfusion has started is effective in reducing infarct volume (by 50–90%) and reducing animal mortality [74, 75]. Moreover, hypothermia exerts its neuroprotective effects partly by suppressing the BBB opening [76, 77] and by attenuating neutrophil infiltration in the ischemic penumbra [78, 79]. Hence, hypothermia exerts at least some of its neuroprotective effects by modulating mechanisms responsible for reperfusion injury.
In most clinical studies, hypothermia is induced by surface cooling with the use of cooling blankets, alcohol applied to exposed skin, or ice bags to groin, axilla and neck. Although whole-body surface cooling is the simplest and most cost-effective option for inducing hypothermia [80], it has two major drawbacks. First, it takes several hours to reach the target body core temperature. All studies report a 3- to 7-hour time period for cooling down to 32–34°C [81–84]. Target temperature should be reached as soon as possible to prevent reperfusion injury; the 3–7 hour time period to reach target temperature is too long and the therapeutic window will be missed for many patients. Theoretical models predict that intracerebral hypothermia can be achieved up to 30 times faster than whole-body hypothermia [85]. The second drawback of whole-body cooling is the high incidence of complications such as pneumonia, arrhythmias/bradycardias, hypotension and coagulopathies [81–83, 86, 87]. Selective brain cooling may dramatically improve the safety profile of therapeutic hypothermia. Selective brain cooling can be achieved by means of an endovascular catheter that infuses the cerebral vasculature with cold saline. A recent study examined whether cold saline (20°C) infused intra-arterially into the ischemic territory of rats that had undergone MCA occlusion could selectively induce cerebral hypothermia and reduce brain injury from stroke [88]. Following cold saline infusion, cerebral temperature fell within 1 min to 33.4°C, while rectal temperature was maintained above 36°C at all times. A significantly reduced infarct volume was found in ischemic rats that received a local cooling infusion compared with control animals (Fig. 3). Further clinical research is needed to explore the therapeutic potential of selective brain cooling and the yet-unknown adverse effects, as well as to establish the extent to which selective brain cooling specifically modulates reperfusion injury.
Fig. 3Graph showing percentages of infarct volume in the four ischemic rat groups (stroke, stroke with local infusion of saline at 20°C and 37°C, and stroke with systemic infusion at 20°C). A significantly (P < 0.001) reduced infarct volume was found in ischemic rats that received a local cooling infusion compared with the rats in the other three groups (reproduced with permission from Ding et al. [88])
Conditioned blood reperfusion
Conditioned blood reperfusion is a therapeutic strategy in which the ischemic organ is perfused with autologous conditioned blood at the onset of reperfusion. Blood conditioned by filtration is depleted of leukocytes and platelets, both at levels far greater than 99.99%, and the complement cascade is modulated in such a way as to prevent the deposition of MAC onto ischemic tissue [89]. Filtering the reperfused blood in this manner may reduce further tissue damage after restoration of circulation [89, 90]. Blood can be filtered using the conditioned blood reperfusion application (CoBRA) filter (Pall B-1328 filter; Pall Corporation, East Hill, N.Y.) at the onset of reperfusion [89]. Withdrawn arterial blood can be filtered through the CoBRA filter and returned back to the cerebral circulation via an endovascular catheter. A small feasibility and safety clinical trial of blood filtering during primary percutaneous transluminal coronary angioplasty for acute myocardial infarction demonstrated that integration of the CoBRA filter into an active hemoperfusion system did not delay the procedure or cause any complications [89]. Furthermore, conditioned blood reperfusion resulted in an acute improvement in ventricular function. Animal models of hypothermic circulatory arrest have demonstrated improvement of neurological outcomes following conditioned blood reperfusion [91–93]. In one of these studies, pigs received blood filtered using the CoBRA filter [92]. Each pig received a common carotid artery filtered perfusion for 10 minutes. Pigs receiving conditioned perfusion had significantly better neurological scores than control pigs (P < 0.005). These findings suggest that multifocal intervention aimed at leukocytes, platelets and complement attenuation can dramatically improve neurological recovery from ischemic insult. Future studies will establish the efficacy of this strategy and the incidence of possible adverse effects, such as coagulapathies and infections.
Further studies are needed to evaluate the role of local hypothermia and conditioned blood perfusion in reducing postischemic reperfusion injury. It is possible, yet still speculative, that in the future interventional neuroradiologists may use these approaches following recanalization with thrombolytic agents or mechanical embolectomy to lessen reperfusion injury.

reperfusion injury
cerebral ischemia
magnetic resonance imaging
hypothermia
conditioned blood reperfusion


Optimisation of Biochemical Condition and Substrates In Vitro for Tissue Engineering of Ligament

In this work, we analysed the effect of growth factors on in vitro cell proliferation and collagens synthesis by fibroblasts cultured for 72 h on different substrates (silicon sheet with or without 1% gelatin, and glass as control surface) for ligament tissue engineering. A human fibroblast cell line (CRL-2703) was used. The synthesis of type I and type III collagens were evaluated qualitatively and quantitatively by RT-PCR and confocal microscopy, respectively. Cell proliferation was evaluated by two methods: (1) MTT assay (2) cell cycle analysis. It was found that PDGF-AB stimulate the proliferation of fibroblast cultured on gelatin coated silicon sheet in dose dependant manner with a maximum effect at 10 ng ml−1. The exogenous TGF-β1 induced the expression of type I and type III collagens in a dose and substrate-dependant manner. We deduce from this work that biochemical conditions and substrates have an important impact for optimisation of the tissue neo synthesis.
INTRODUCTION
Tissue engineering is a multidisciplinary field at the crossroads of biochemistry, mechanics, engineering and materials science to develop substitutes for replacing injured or diseased tissues.14,15,25,48 For all applications, it is necessary to take into account of several points: cell source, growth factors, mechanical forces, substrates, and the behaviour of the cells on the substrate.
Substrates or scaffolds are defined as the matrix that provides a suitable environment for cell proliferation and differentiation. Some scaffolds have been prepared from natural and synthetic materials, such as collagen, poly (glycolic acid) (PGA), and copolymers of l-lactic acid and є-caprolactone. It is necessary to improve the physical and chemical nature of scaffold for the superior induction of tissue regeneration. However, the chemical composition of the scaffold can have great influence on its biocompatibility.5,38 The source, solubility, degree of cross linking, physical form of the protein coating substrates, and the substrates itself can influence the cell functions like proliferation and metabolic activity.5
Cells and materials are two essential components in ligament tissue engineering. Materials could interfere with cells adhesion, proliferation, and differentiation. Currently, there are two common approaches to modify the materials to improve their biocompatibility, coating with biocompatible materials, or surface modification. As nearly all interaction between cells and artificial surfaces are mediated by a layer of absorbed protein, many proteins have been coated on the surface of target materials to promote adhesion, (i.e. fibrinogen, collagen, hyaluronan,...).13 Protein coated substrates are provided as a two dimensional substrates. Moreover, it has been shown that fibroblast attachment, morphology, proliferation, and collagen synthesis varied from different culture substrates and the type of the fibroblast obtained from different organs/tissue.9
An appropriate scaffold for ligament regeneration must be able to provide mechanical strength to initially withstand the in vivo force and degrade safely at an appropriate rate in vivo.39In vitro studies, a planar artificial substrate (i.e. silicon membrane) is usually used for growing cells and subjected them to mechanical loading.
Furnishing scaffolds with biochemical factors are also promising. Many biochemical factors including growth factors could promote tissue regeneration and/or healing processes.13
Growth factors are known to play an important role in the healing response of connective tissues.27 Several reports indicate the significance of growth factors in the intrinsic healing process of injured ligaments.24,32 Many growth factors affect the tendon and ligament healing process after injury by stimulating extra-cellular matrix (ECM) synthesis and cell proliferation.3,17,21,47 It has been shown that exogenous Transforming Growth Factors β1 (TGF-β1) increased the collagen synthesis of human anterior cruciate ligament cells and human ligamentum flavum.22,35 Platelet Derived Growth Factor-AB (PDGF-AB), one of the major serum growth factor, is known to enhance cell proliferation and healing responses,26,46 as well as to increase rupture force, stiffness and breaking energy of the tissue.31
The definition of the optimal condition of culture, such as coating of the substrate, synergistic effect of the substrate and growth factors are required for ligament tissue engineering.
In order to define the optimal concentration of growth factors and the evolution of cells culture on silicon membrane, which will be subjected to cyclic stretch for later mechanical study, the objective of the present work was to investigate the influences of individual growth factors and substrates on the cell proliferation and ECM synthesis. In this work, we studied the cell viability, proliferation, collagen production of fibroblast seeded on the different substrates and the effect of PDGF-AB on the cell proliferation and TGF-β on the ECM synthesis to allow the optimisation of culture conditions, which could be used for in vitro reconstruction of ligament/tendon tissues.
MATERIALS AND METHODS
Cells and Culture Conditions
Human skin fibroblast cell line (CRL-2703) was purchased from American Type Culture Collection (ATCC, No. 2584882, Manassas, VA). The cells were placed in 75 cm2 plastic culture flask with Dulbecco’s modified Eagle’s medium/Nutrient F-12 (GibcoBRL, Life Technologie, France) containing 10% decomplemented foetal bovine serum (FBS) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM l-glutamine, 2,5 μg/ml fungizone, 1% non-essential amino acids (GibcoBRL, Life Technologie, France), 150 μg/ml l-ascorbic acid, 1.5 g/l NaHCO3 (Sigma, France) and were incubated at 37°C [air (95%) and CO2 (5%)]. The medium was changed every 2 days. At confluence, cells were detached from the culture flask with 0.125% trypsin–EDTA mixture (Sigma, France) for 10 min at 37°C, and then resuspended in complete medium until confluence.
Substrate Surface Treatment
Transparent, elastic silicon sheet (thickness: 0.0254 cm) (Specialty manufacturing INC, USA) was punched out into round sheets (diameter: 1.4 cm). Glass slips (diameter: 1.4 cm) (Polylabo, France) and silicon sheets were sterilised by overnight immersion in 75% ethanol aqueous solution, air- dried, then placed in a 24-well multiwell culture plate, and sterilised by 10 min UV irradiation. Gelatin solutions (1% w/v in distilled water) (Sigma, France) were sterilised by filtration before use. Cells were seeded into 24 wells culture plates at a density of 3 × 104 cells/cm2 on different surface glass slips (control surface), silicon sheet, coated or not with gelatin for 24 h at 37°C to allow cell attachment.
Biochemical Stimuli
The concentrations of growth factors used in this study included those noted to be effective in previous studies.37,42 The concentration range for TGF-β was 0.12–25 ng ml−1 and from 1 to 100 ng ml−1 for PDGF-AB. In our study, five concentrations of TGF-β1 (0.1, 0.5, 1, 5, 10 ng ml−1) and four concentrations for PDGF-AB (1, 5, 10 and 50 ng ml−1) were used. These concentrations were added to the cultures in order to determine their effects on the cell proliferation for PDGF-AB and collagen production for TGF-β1. Cell proliferation and collagen synthesis were determined 72 h after the addition of the growth factors. Culture medium containing 10%FBS without growth factors was used as control.
Fibroblast Proliferation Experiments
MTT Assay
After 72 h PDGF-AB treatment, MTT 3-[4,5-Dimethylthiazol-2,5-diphenyltetrazolium bromide, blue Thiazolyl] (Sigma, France) was prepared in PBS at a final concentration of 2 mg/ml. Then 125 μl of the solution were added to each well containing 500 μl of DMEM without red phenol. The cells were incubated for 4 h at 37°C to allow the yellow dye to be transformed into blue formazan crystals by the mitochondrial dehydrogenases. The insoluble product was dissolved by addition of 800 μl of acidified isopropanol (0.04 N HCl in isopropanol), and thoroughly mixed to dissolve the dark blue crystals. The supernatants were removed, centrifuged, and read within 30 min by using a Beckman DU-600 spectrophotometer (USA) with a wavelength of 570 and 700 nm was used as reference wavelength. The optical density difference OD = OD570 nm−OD700 nm was estimated.
Cell Cycle Analysis
After 72 h PDGF-AB treatment, cells were trypsinisation then collected by centrifugation (500g) for 10 min. Cells pellets were washed twice with 2 ml of PBS. 50 μl of the washed cell suspension (15 × 104 cells) were stained according the kit instruction (Coulter DNA Prep Reagents kit; Beckman-Coulter, Miami, USA) in order to analyse the cell cycle by flow cytometry (EPICS XL Coulter; Beckman-Coulter). Primary histograms were generated by gating the cells according to the surface and the peak of the fluorescence signal in order to avoid taking doublets into account. These histograms were then submitted to computerised calculations of the cell cycle by using polynomial analysis derived from the Dean and Jett model Multi Cycle AV (Phoenix Flow System, San Diego, USA). For each testing condition, the percentage of cells in each phase was determined. DNA content was analysed in at least 105 nuclei in each sample.
RNA Isolation and RT-PCR
Total cellular RNA were isolated from fibroblasts after dissolution in TRIzol reagent (Invitrogen, France) according to the manufacturer’s instructions. RNA were extracted using chloroform, then samples were centrifuged (9500g) for 15 min at 4°C. Isopropanol was added to precipitate the RNA and the samples were centrifuged for 10 min under the same conditions. The RNA pellet was washed by addition of 1 ml of 75% ethanol and centrifuged at 5600g for 5 min. The pellet was resuspended in 50 μl DEPC-treated water. The concentrations of RNA were determined spectrophotometrically at 260 nm and the purity of RNA were controlled by the ratio DO260 nm/DO280 nm. The first strand cDNA was prepared from the RNA with reverse transcriptase (kit ThermoScript RT-PCR System) (BioRad, USA). The cDNA was amplified by polymerase chain reaction (Platinum Taq DNA Polymerase) (Invitrogen, France) using the primers shown in Table 1. Gel electrophoresis of RT-PCR products was performed in a 2% agarose (Sigma, France) run in 0.5× Tris/-boric acid EDTA (TBE) buffer (Bio-Rad, USA). The gel was stained in a 0.5 μg/ml ethidium bromide solution for 20 min and the images were obtained using a Bio-Rad GS-2000 Molecular Imaging System. Bands intensities were evaluated against background quantified by Quantity One Image software (Bio-Rad, USA) and normalized to β-actin.
TABLE 1.Prime sequence for RT-PCR.MRNAPrimer sequenceProduct size (bp)Type I collagen5′-TCC CCA GCC ACA AAG AGT CTA CA-3′1555′-GTG ATT GGT GGG ATG TCT TCG TC-3′Type III collagen5′-CTG CCA TCC TGA ACT CAA GAG TGG-3′4475′-CCA TCC TCC AGA ACT GTG TAG G-3′Β-actin5′-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3′8385′-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3′
Immuonofluorescence Labelling
After incubation with TGF-β1, the cells were rinsed with PBS. The cells were incubated for 45 min with anti-collagen I (1/50 dilution) or anti-collagen III (1/100 dilution) rabbit IgG polyclonal (Calbiochem, France) in DMEM without red phenol (GibcoBRL, Life Technologie, France) containing 0.5% (w/v) BSA. The cells were then rinsed thoroughly in DMEM without red phenol and incubated with the second antibody, Alexa™-488 conjugated goat anti-rabbit IgG (λex = 488 nm, λem = 515 nm) (1/200 dilution) for 30 min in the dark. After washing with DMEM without red phenol, the cells were fixed with 1% para-formaldehyde (Sigma, France) in PBS for 10 min, and rinsed in PBS. The cells were observed under a fluorescence confocal microscope (SP2-Leica micro systems, Germany).
Statistical Analysis
Differences in cell proliferation and collagen synthesis were analysed by one-way ANOVA analysis to evaluate differences between groups with PLSD Fisher correction (Statview IV®, Abacus Concepts Inc, Berkley,CA, USA). A p value less than 0.05 was considered statistically significant. All data were expressed as the mean ± standard deviation (SD).
RESULTS
All the works were realized on three types of substrates (silicon sheet, silicon sheet with 1% gelatin coating as testing surfaces and glass surface used as a control).
Effect of PDGF-AB on Cell Proliferation
We studied the effect of PDGF-AB on fibroblast proliferation according to substrates and PDGF concentrations by two different and complementary methods.
MTT Assays
The results of the MTT assay are summarized in Fig. 1. These results were obtained from fibroblast cultured on different substrates at day 3 in the presence or absence of four different concentrations of PDGF-AB. On the glass and gelatin coated silicon sheet cultures, PDGF-AB induced a dose-dependent increase in the value of A570. On glass culture, the significant increase was observed in the range of 5–50 ng ml−1 PDGF-AB (p < 0.01). On the gelatin coated silicon sheet culture, the significant increase was observed in the range of 1–50 ng ml−1 PDGF-AB (p < 0.01). On the silicon sheet culture, the values of A570 decrease when the concentration of PDGF-AB was above 5 ng ml−1.
FIGURE 1.Dose dependent effect of PDGF-AB on cell proliferation by MTT assays. Cells were treated with different concentrations of PDGF-AB for 72 h. The metabolic activity of fibroblast seeded on glass, silicon sheet with or without 1% gelatin was measured by MTT assays. # p < 0.05 versus control glass, *p < 0.05 versus control silicon sheet, ¤ p < 0.05 versus control gelatin coated silicon sheet, n = 12.
Nevertheless, we observed a loss of cells on silicon sheet at 50 ng ml−1 PDGF-AB.
Cell Cycle Analysis
To confirm the MTT results, we also studied the effect of PDGF-AB on cell cycle (Figs. 2 and 3). Cell cycle analysis was performed on day 3 of the culture. It was observed in the three types of substrates that PDGF-AB induced a concentration-dependant decrease in the percentage of cells in G0/G1 phase and an increase in S and G2/M phase.
FIGURE 2.Cell cycle analysis by flow cytometry. Representative cell cycle histogram of the cells cultured on glass (a), gelatin coated silicon sheet (b). After 3 days of treatment with PDGF-AB (1, 5, 10, 50 ng ml−1), cells were recovered and DNA staining with propidium iodide (IP) was performed. Ten thousand cells were analysed for each sample, n = 9.FIGURE 3.Effect of different concentrations of PDGF-AB on fibroblast cell cycle regulation. Cells cultured on glass (a), silicon sheet (b), gelatin coated silicon sheet (c). Cells were subjected to different concentrations of PDGF-AB (1, 5, 10, 50 ng ml−1). The percentage of cells in each phase of the cell cycle was analysed by flow cytometry. Results are represented as mean ± SD of three independent experiments for each substrate. p < 0.05 versus control, n = 9.
On gelatin coated silicon sheet, the percentage of cells in S-phase was higher than that without gelatin or glass (Fig. 3C). Moreover, cells exposed to 10 ng ml−1 of PDGF-AB had the highest percentage of cells in G2/M phase (p < 0.01).
Effect of TGF-β1 on the Expression of Collagens
Effect of TGF-β1 on Type I and Type III Collagens mRNA Expressions
The gene expressions of collagens in cells cultured on different substrates were measured by RT-PCR. RT-PCR analysis of type I and type III collagen were performed at day 3 in the presence or absence of five different concentrations of TGF-β1. The RT-PCR results on human fibroblasts seeded on the three substrates are shown in Fig. 4. The quantitative results concerning type I and type III collagen are summarised in Fig. 5.
FIGURE 4.Effect of different concentrations of TGF-β1 on the mRNA expression of type I and type III collagen. Representative RT-PCR results of human fibroblasts seeded on glass (a), silicon sheet (b), gelatin coated silicon sheet (c). The OD of each band was normalised to the values of housekeeping gene, β-actin. The cells were cultured on different substrates in the presence of different concentrations of TGF-β1 (0.1, 0.5, 1, 5, 10 ng ml−1) for 3 days.FIGURE 5.Quantitative measurement of the RT-PCR results. a: type I collagen, b: type III collagen. Results are expressed as mean ± SD of a representative of three different experiments carried out in duplicate. Black bars represent cell culture on glass; bright grey bars represent cell culture on silicon sheet; dark grey bars represent cell culture on gelatin coated silicon sheet. # p < 0.05 versus control glass, *p < 0.05 versus control silicon sheet, ¤ p < 0.05 versus control gelatin coated silicon sheet, n = 6.
The mRNA expression of collagen type I is shown in Fig. 5a. On the glass culture and gelatin coated silicon sheet cultures, there was no significant difference in the mRNA expression of collagen type I between the TGF-β1 treated group and the control group. Otherwise, on the silicon sheet culture, the mRNA expression of collagen type I increased significantly in the presence of 0.1 ng ml−1 TGF-β1.
The mRNA expression of collagen type III is shown in Fig. 5b. On the glass culture, we can see a cyclic effect of TGF-β1 on the expression of collagen type III and it reached a maximum response at 10 ng ml−1 TGF-β. Also, compared to control group, the mRNA expression of collagen type III was decreased significantly at a concentration of 0.5 and 1 ng ml−1 TGF-β1 (p < 0.05).
On the gelatin coated silicon sheet cultures, the significant increase in the mRNA expression of collagen type III was observed by the addition of 0.1 and 10 ng ml−1 TGF-β1 (p < 0.05).
On the silicon sheet culture, the mRNA expression of collagen type III decrease when the concentration of TGF-β1was above 0.5 ng ml−1.
Qualitative Expression of Type I and Type III Collagens
In parallel of the RT-PCR experiments, we carried out some experiments by confocal microscopy to visualise the expression of collagen. The protein expression of collagen type I and type III of the cells cultured on glass were revealed by indirect immunofluorescence staining. As shown in Fig. 6, collagen type I occurred as spots dispersed around the cells, and collagen type III was organised as network-like fibre. There is no difference in the expression of type I collagen with the different concentration of TGF-β1. In contrast, the expression of type III collagen was increased after the addition of TGF-β1. The same expression model of collagens was observed on the other substrates (data not shown).
FIGURE 6.Immunofluorescence staining of collagen in monolayer cultures of fibroblast on glass visualised by confocal microscopy. (A) Type I collagen is identified on the 3rd day of culture by use of anti collagen type I antibodies. Only spots of this type of collagen were secreted by the cells (arrowhead). (B) Type III collagen is identified by use of anti collagen type III antibodies. It appears like network in the cell (arrow). (objectif 40×/NA 0.80, Leica, Germany). Bar 40 μm. (a): Control cell (DMEM + 10%FBS); (b): Cell treated with 0.5 ng ml−1 TGF-β1; (c): Cell treated with 10 ng ml−1 TGF-β1.
DISCUSSION
As cell proliferation and matrix synthesis are important parameters in tissue engineering, the aim of this study was to optimise the in vitro culture conditions on ligament/tendon tissues for future experiments in bioreactors. The effect of PDGF-AB and TGF-β1 on the cell proliferation and matrix synthesis of fibroblast cultured on three different types of substrates (silicon sheet with or without 1% gelatin, glass) for 3 days were investigated in this study. For the tissue regeneration, it is better to use three-dimensional scaffolds to investigate the effects of the properties of the material on cell behaviour. However, there are many parameters of the scaffolds to be considered, such as porosity, pore size, pore interconnectivity, surface property, topography and chemical composition. Therefore, in this study, two-dimensional substrates were used to investigate the influence of substrate surface property on the proliferation and ECM synthesis of fibroblast.
Firstly, we used PDGF-AB, which was widely used as a mitogenic growth factor and as a chemotactic agent for cells such as fibroblasts.45 Our experiments demonstrated that cell proliferation was enhanced by PDGF-AB. This result was consistent with those of the previous studies on lung fibroblast,6,30 canine anterior cruciate ligament cell,8 human periodontal ligament (PDL) cells36 and human anterior cruciate ligament cell (ACL).33 PDGF increased the number of fibroblasts available to enter the cell replication cycle by stimulating termination of the G0 phase of the cell cycle. The results obtained in this study are similar to those reported by Letson27 and Schmidt46 which showed that PDGF would be an effective growth factor that stimulates resting cells to advance from G0 to G1 of the cell cycle.
Although the addition of PDGF-AB resulted in significant increase in cell proliferation on all the substrates, the highest increase was found on gelatin coated silicon sheet culture. This result may be explained by the physical properties of the substrate surface. High cell proliferation was observed for the substrates with contact angles of 60–80° as well as on protein coated culture plates such as gelatin.18 Park et al. have demonstrated that mescenchymal stem cells (MSCs) cultured on collagen I or gelatin coated membranes expressed similar levels of smooth muscles (SM) markers.41 Hori et al. reported that the morphology and the proliferation of MSCs were influenced by the type of substrate and culture medium.18 The use of gelatin coated silicon sheet as a substrate allows to enhance the cell adhesion, the differentiation and the proliferation.
In addition, the higher response was found to 10 ng ml−1 PDGF on gelatin coated silicon sheet culture but not to the maximal dose as 50 ng ml−1 PDGF-AB. This finding indicates that it exist a dose-dependant effect of PDGF-AB on cell proliferation in this culture model and 10 ng ml−1 PDGF might be the optimal concentration. Indeed, our result was consisting with the previous experiment which demonstrated that 10 ng ml−1 of PDGF promoted fibroblast attachment to collagen coated plates and proliferation.21,36
Secondly, we investigated the effect of TGF-β1 on collagen synthesis. TGF-β1 is the prototype of a large family of growth factors regulating cell growth, development, differentiation, immune system and tissue repair. On the one hand, TGF-β1 mostly inhibited the cell growth both in vivo and in vitro.2 It could arrests cell cycle progression at a point in late G1 phase.12,19,43 Furthermore, stimulatory effects of PDGF-AB have been demonstrated to be blocked by the presence of TGF-β1.20,23 On the other hand, it has been shown that TGF-β regulates production of the ECM, including collagen, fibronectin and tenascin.11,16,40,44
Therefore, in the second step of our works, we study the effect of TGF-β1 on the ECM synthesis. Our data showed that the exogenous TGF-β1 influenced the expression of type I and type III collagen in a substrate- dependant manner.
In our study, the mRNA expression of collagen type III was higher than that of collagen type I when the cells were cultured on glass and gelatin coated silicon sheet for 72 h. During the early phases of ligament healing and remodelling more type III collagen is produced than type I collagen.1,10,49 Therefore the greater upregulation of type III collagen as compared to type I collagen is a positive indicator that the cells are secreting signals for the healing and remodelling process.
However, on glass, we observed the highest expression of type III collagen mRNA in the control group. In the present work, we used culture medium containing 10% FBS. Although, we did not add any TGF-β1 in the medium, it seemed that this increase was due to the presence of growth factors in the serum and which normally stimulate protein synthesis and other different biological processes.
Furthermore, we found that TGF-β1 effected the expression of type I and type III collagen synthesis by fibroblasts with a dose dependant manner. The expression of type III collagen mRNA on gelatin coated silicon sheet was significantly increased at 0.1 and 10 ng ml−1 TGF-β1. The results of this study are consistent with the previous work which demonstrated that 10 ng ml−1 of TGF-β1 may promote fibroblast attachment.21 In 2D cell culture, TGF-β1 has been demonstrated to have a dose-dependent effect on collagen production.4,7,28,29,32,34
In summary, our results demonstrated that PDGF-AB promoted the cell proliferation, which is a basic step in the process of ligament repair. Our data also suggest that TGF-β1 is an important cytokine during the early phases of ligament healing process for its role in the promotion of extra cellular matrix formation. These results allowed us to determine the main biochemical conditions of in vitro construction of ligament/tendon tissues.
Our results implied that it is better to use protein-coating substrate to improve the proliferation and the synthesis of collagen for later mechanical stretching study. Further studies are needed to determine the combination of these two growth factors and their effect on the biological processes.

substrates
growth factors
cell proliferation
collagens synthesis
fibroblasts