RESUMO
BACKGROUND: Baló's Concentric Sclerosis (BCS) is a rare heterogeneous demyelinating disease with a variety of phenotypes on Magnetic Resonance Imaging (MRI). Existing literature lacks data especially on the therapeutic approach of the disease which we intended to elucidate by means of suggesting a new possible BCS classification and introducing different therapeutic concepts based on each BCS-subgroup characteristics. METHODS: We present a retrospective study of eight treated patients with BCS-type lesions, emphasizing on MRI characteristics and differences on therapeutic maneuvers. RESULTS: Data analysis showed: at disease onset the BCS-type lesion was tumefactive (size ≥2 cm) in 6 patients, with a mean size of 2.7 cm (± 0.80 SD); a coexistence of MS-like plaques on brain MRI was identified in 7 patients of our cohort. The mean age was 26.3 years (±7.3 SD) at disease onset and the mean follow-up period was 56.8 months (range 9-132 months). According to radiological characteristics and response to therapies, we further categorized them into 3 subgroups: a) Group-1; BCS with or without coexisting nonspecific white matter lesions; poor response to intravenous methylprednisolone (IVMP); treated with high doses of immunosuppressive agents (4 patients), b) Group-2; BCS with typical MS lesions; good response to IVMP; treated with MS-disease modifying therapies (2 patients), c) Group-3; BCS with typical MS lesions; poor response to IVMP; treated with rituximab (2 patients). CONCLUSIONS: Our study introduces a new insight regarding the categorization of BCS into three subgroups depending on radiological features at onset and during the course of the disease, in combination with the response to different immunotherapies. Immunosuppressive agents such as cyclophosphamide are usually effective in BCS. However, therapeutic alternatives like anti-CD20 monoclonal antibodies or more classical disease-modifying MS therapies can be considered when BCS has also mixed lesions similar to MS. Future studies with a larger sample size are necessary to further establish these findings, thus leading to better treatment algorithms and improved clinical outcomes.
Assuntos
Esclerose Cerebral Difusa de Schilder/tratamento farmacológico , Imageamento por Ressonância Magnética , Metilprednisolona/uso terapêutico , Adolescente , Adulto , Encéfalo/patologia , Estudos de Coortes , Esclerose Cerebral Difusa de Schilder/patologia , Feminino , Humanos , Masculino , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Alemtuzumab has been demonstrated to reduce the risks of relapse and accumulation of sustained disability in Multiple Sclerosis (MS) patients compared to ß-interferon. It acts against CD52, leading primarily to lymphopenia. Recent data have shown that mild neutropenia is observed in 16% of treated MS-patients whereas severe neutropenia occurred in 0.6%. CASE PRESENTATION: Herein, we present the case of a 34-year-old woman with relapsing-remitting MS, with a history of treatment with glatiramer acetate and natalizumab, who subsequently received Alemtuzumab (12 mg / 24 h × 5 days). 70-days after the last Alemtuzumab administration, the patient displayed neutropenia (500 neutrophils/µL) with virtual absence of B-cells (0.6% of total lymphocytes), low values of CD4-T-cells (6.6%) and predominance of CD8-T-cells (48%) and NK-cells (47%); while large granular lymphocytes (LGL) predominated in the blood-smear examination. Due to prolonged neutropenia (5-days) the patient was placed on low-dose corticosteroids leading to sustained remission. CONCLUSION: This is the first case of a patient with relapsing-remitting MS with neutropenia two months post-Alemtuzumab, with simultaneous presence of LGL cells in the blood and a robust therapeutic response to prednisolone. We recommend testing with a complete blood count every 15 days in the first 3 months after the 1st Alemtuzumab administration and searching for large granular lymphocytes cell expansion on microscopic examination of the peripheral blood if neutropenia develops.
Assuntos
Alemtuzumab/efeitos adversos , Fatores Imunológicos/efeitos adversos , Linfócitos/patologia , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Neutropenia/induzido quimicamente , Adulto , Anticorpos Monoclonais Humanizados/uso terapêutico , Feminino , HumanosRESUMO
OBJECTIVES: Myotonic dystrophy type 1 is associated with various oculomotor, vestibular, and auditory abnormalities. However, auditory system investigation has been mainly performed with the subjective method of pure-tone audiometry. In this study, a detailed vestibular and audiological evaluation was undertaken, including the objective and more sensitive method of transiently evoked otoacoustic emissions (TEOAEs). MATERIALS AND METHODS: Twenty-four patients with genetically diagnosed myotonic dystrophy type 1 and 21 controls were studied. Audiological and vestibular investigations included pure-tone audiometry, tympanometry, auditory brainstem responses (ABRs), TEOAEs, and electronystagmography. RESULTS: Hearing impairment was evident in 15 (62.5%) patients and in nine of them (37.5%) ABR abnormalities were found. However, subclinical cochlear damage was found in all patients, as evidenced by absent emissions or lower otoacoustic emission amplitude. Vestibular hypesthesia was found in nine patients (37.5%), accompanied by spontaneous nystagmus in four of them (15.6%). CONCLUSIONS: Auditory and vestibular abnormalities are quite common in patients with myotonic dystrophy type 1. However, it appears that subclinical cochlear damage is an ubiquitous finding of the disease.
Assuntos
Orelha Interna/fisiopatologia , Perda Auditiva Neurossensorial/etiologia , Distrofia Miotônica/fisiopatologia , Nistagmo Patológico/etiologia , Testes de Impedância Acústica , Adulto , Audiometria de Tons Puros , Cóclea/fisiopatologia , Eletronistagmografia , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Hipestesia/etiologia , Hipestesia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Nistagmo Patológico/fisiopatologia , Emissões Otoacústicas Espontâneas , Prevalência , Vestíbulo do Labirinto/fisiopatologiaRESUMO
BACKGROUND/AIMS: The onset of multiple sclerosis (MS) in Greece has not been systematically studied. We sought to provide data on the onset of MS in Greece with detailed information regarding initial symptoms, and to confirm the prognostic significance of demographic and clinical factors at onset. METHODS: We studied 1,034 consecutive patients with MS and independently assessed 265 patients 'seen at onset'. We used the MS severity score and survival analysis (time to reach an Expanded Disability Status Scale score of 4.0) to evaluate the prognostic significance of factors at onset. RESULTS: Female-to-male ratio was 1.9:1 and mean age at onset was 30.7 +/- 9.9 years. MS was primary progressive in 9.6%. Initial symptoms were optic neuritis in 20.1%, brainstem dysfunction in 14.7%, dysfunction of long tracts in 49.3%, cerebral dysfunction in 1% and a combination of symptoms in 14.9%. In 'seen at onset' patients, detailed data on initial symptoms are presented. Female gender, earlier age at onset, 'bout onset' and onset with optic neuritis were associated with less severe disease and longer time to disability. CONCLUSION: The onset of MS in Greece is similar to Western populations. Initial symptoms are within the expected spectrum. Prognostic significance of factors at onset is as previously identified.
Assuntos
Esclerose Múltipla , Adulto , Idade de Início , Feminino , Grécia/epidemiologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/epidemiologia , Esclerose Múltipla/fisiopatologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Estatísticas não Paramétricas , Adulto JovemRESUMO
Spinal and bulbar muscular atrophy (Kennedy's disease) has been associated with balance dysfunction and falls. However, postural control has not been studied quantitatively. Here, we quantified upright stance and aimed to disentangle the role of vestibular, proprioceptive and oculomotor deficits. Static balance was assessed in Kennedy patients (n = 7) during quiet stance on a force platform under different visual and proprioceptive feedback conditions. Vestibular function was assessed with the video head impulse test. Sural nerve neurography was employed to evaluate the severity of peripheral neuropathy. Also, horizontal saccades were recorded and quantified by the main sequence relationship. Posturographic analyses revealed significantly increased body sway, more pronounced in conditions with closed eyes, which was also reflected in the calculated Romberg indices. Horizontal vestibulo-ocular reflex gains were normal, i.e. > 0.75. In contrast, compound sensory nerve action potentials were markedly decreased in all patients (mean = 2.4 µV). Two patients showed slow saccades with increased exponential main sequence constants. We conclude that Kennedy patients exhibit severe deficits in quiet stance. Postural instability is greatest in conditions of absent vision with reduced proprioception being the main determinant of unsteadiness. Some patients show slowed saccadic eye movements suggesting a nuclear abducens neuronopathy.
Assuntos
Atrofia Bulboespinal Ligada ao X/fisiopatologia , Equilíbrio Postural/fisiologia , Propriocepção/fisiologia , Movimentos Sacádicos/fisiologia , Doenças Vestibulares/fisiopatologia , Potenciais de Ação/fisiologia , Adulto , Estudos de Casos e Controles , Humanos , Masculino , Pessoa de Meia-Idade , Células Receptoras Sensoriais/fisiologia , Nervo Sural/fisiologiaAssuntos
Doença de Charcot-Marie-Tooth/genética , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteínas da Mielina/genética , Proteínas de Neurofilamentos/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Análise Mutacional de DNA/métodos , Feminino , Grécia , Humanos , Masculino , Adulto JovemRESUMO
Neuromyelitis optinca (NMO) represents a serious demyelinating disease of the central nervous system selectively attacking the spinal cord and optic nerve. Early differential diagnosis from multiple sclerosis is of vital importance, as NMO mandates immunosuppressive and not immunomodulatory treatment. Rituximab has been recently introduced as a treatment option for NMO. However, optimal surrogate measures and treatment intervals are still unclear. Five patients (females, mean age 54±10.21years) with NMO and NMO spectrum disorders (NMOSD) were evaluated with respect to disability and relapse rate. All patients were found positive for NMO IgG. All patients (three with NMO and two with NMOSD, 1 patient with recurrent optic neuritis and 1 patient with recurrent myelitis) had received rituximab treatment for six years. One patient with NMOSD received cyclophosphamide prior to rituximab while two were misdiagnosed as multiple sclerosis and had received interferon treatment. All received rituximab infusion of 375mg/m2 once per week for 4weeks and then every two months for the first two years and then every six months. B-cell counts were measured every two months and were kept in almost undetectable levels. No relapse was noted during the treatment period while EDSS score was improved in all patients. No severe adverse effects occurred during RTX treatment. Rituximab treatment on NMO and NMOSD patients showed significant improvement in disability and relapse-rate without any significant adverse effects.
Assuntos
Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Fatores Imunológicos/uso terapêutico , Neuromielite Óptica/tratamento farmacológico , Neuromielite Óptica/patologia , Rituximab/uso terapêutico , Idoso , Autoanticorpos/sangue , Linfócitos B/efeitos dos fármacos , Avaliação da Deficiência , Feminino , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Nervo Óptico/diagnóstico por imagem , Estudos Retrospectivos , Medula Espinal/diagnóstico por imagemAssuntos
Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Proteína P0 da Mielina/genética , Proteínas da Mielina/genética , Doença de Charcot-Marie-Tooth/epidemiologia , Análise Mutacional de DNA , Duplicação Gênica , Grécia/epidemiologia , Humanos , Proteína beta-1 de Junções ComunicantesRESUMO
OBJECTIVE: Neurotransmitter systems participate in the regulation of food intake, and their activities are expected to influence eating behavior. DESIGN AND METHODS: We investigated possible associations between body mass index (BMI) and central noradrenaline, serotonin, and dopamine activities, as reflected by the cerebrospinal fluid levels of their main metabolites methoxyhydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA), respectively. We studied 192 subjects (111 males, 81 females) admitted to neurologic clinic for diagnostic investigations that included CSF analysis, and were found not to suffer from any major neurological disease. Subjects were categorized in three groups, namely in lower, in the two middle, and in upper BMI quartiles, the limits calculated separately for males and females. RESULTS: No differences were found in MHPG levels between groups, while subjects in the upper BMI quartile showed significantly elevated levels of 5-HIAA and HVA compared to the levels of subjects in lower and middle quartiles. CONCLUSIONS: The results provide evidence that in overweight subjects there are enhanced demands in serotoninergic and dopaminergic signaling for their reward system that may lead to increased motivation for food consumption. The implication of reward centers in eating behavior supports the hypothesis of common mechanisms in obesity and drug addiction.
Assuntos
Dopamina/líquido cefalorraquidiano , Sobrepeso/líquido cefalorraquidiano , Serotonina/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Índice de Massa Corporal , Feminino , Ácido Homovanílico/líquido cefalorraquidiano , Humanos , Ácido Hidroxi-Indolacético/líquido cefalorraquidiano , Masculino , Metoxi-Hidroxifenilglicol/líquido cefalorraquidiano , Pessoa de Meia-Idade , Norepinefrina/líquido cefalorraquidiano , Análise de Regressão , Adulto JovemRESUMO
Huntington's disease (HD) is an autosomal dominant disorder characterized by a triad of chorea, psychiatric disturbance and cognitive decline. Around 1% of patients with HD-like symptoms lack the causative HD expansion and are considered HD phenocopies. Genetic diseases that can present as HD phenocopies include HD-like syndromes such as HDL1, HDL2 and HDL4 (SCA17), some spinocerebellar ataxias (SCAs) and dentatorubral-pallidoluysian atrophy (DRPLA). In this study we screened a cohort of 21 Greek patients with HD phenocopy syndromes formutations causing HDL2, SCA17, SCA1, SCA2, SCA3,SCA8, SCA12 and DRPLA. Fifteen patients (71%) had a positive family history. We identified one patient (4.8% of the total cohort) with an expansion of 81 combined CTA/CTG repeats at the SCA8 locus. This falls within what is believed to be the high-penetrance allele range. In addition to the classic HD triad, the patient had features of dystonia and oculomotor apraxia. There were no cases of HDL2, SCA17, SCA1, SCA2, SCA3, SCA12 or DRPLA. Given the controversy surrounding the SCA8 expansion, the present finding may be incidental. However, if pathogenic, it broadens the phenotype that may be associated with SCA8 expansions. The absence of any other mutations in our cohort is not surprising, given the low probability of reaching a genetic diagnosis in HD phenocopy patients.
Assuntos
Testes Genéticos , Doença de Huntington/genética , Degenerações Espinocerebelares/genética , Expansão das Repetições de Trinucleotídeos/genética , Adulto , Idoso , Encéfalo/patologia , Feminino , Grécia , Humanos , Doença de Huntington/patologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
A 45-year-old female suffering from severe thoracic pain was admitted to the emergency department of our hospital. Thorough clinical examination revealed paresis of the left lower limb and sensory deficit at the level of the Th4 vertebra. MRI of the thoracic spine demonstrated a lesion at the level of Th1-Th7. Despite initial improvement following i.v. corticosteroid administration, the patient's clinical status deteriorated, with recurrence of myelitis and extension of the lesion to Th12. She developed paraparesis, hyperreflexia and spasticity of both legs, symmetrical sensory deficit below Th4, and sphincter dysfunction. Differential diagnosis included infectious, metabolic, neoplastic/paraneoplastic, and ischemic causes as well as multiple sclerosis. NMO IgG was found positive and led to the diagnosis of longitudinal extensive transverse myelitis (LETM) in the NMO spectrum disorders. Administration of immunosuppressive therapy resulted in gradual improvement of the patient's clinical status and stabilization for five years. In the setting of LETM, patients with antiaquaporin 4 IgGs can present features of coexisting systemic involvement. A thorough differential diagnosis is required to guide appropriate therapy.
Assuntos
Sistema Nervoso Autônomo/fisiopatologia , Síndrome de Brugada/etiologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Eletrocardiografia , Fatores Imunológicos/uso terapêutico , Esclerose Múltipla/complicações , Adulto , Síndrome de Brugada/tratamento farmacológico , Síndrome de Brugada/fisiopatologia , Quimioterapia Combinada , Seguimentos , Humanos , Imageamento por Ressonância Magnética , Masculino , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/tratamento farmacológicoRESUMO
There is considerable evidence that prolactin (PRL) exerts immunomodulatory actions, thus being involved in the processes of autoimmune diseases. Animal studies suggest that elevated serum PRL levels may be related to neuroprotection or participate in remyelination after brain injury. To address this question, we estimated PRL levels in both serum and cerebrospinal fluid (CSF) in drug-free male and female patients with clinically-isolated syndrome (CIS) suggestive of MS (i.e. after the first episode) as well as in patients with relapsing-remitting (RR) MS after two or more relapses, and related them to clinical, paraclinical and laboratory data. Seventy two patients with RR MS and 80 patients with CIS in the age range 17-61 years were studied. PRL levels of patients were compared with 74 control subjects, separately for males and females. Significantly higher PRL levels in serum and CSF were found in female RRMS patients but not in males. Patients with CIS had normal PRL levels. No associations were found with disease activity, duration of illness, presence of active lesions or the presence of oligoclonal bands in CSF. The elevated PRL levels observed in female but not in male RRMS patients, or in patients with CIS, could be suggestive of a sexually dimorphic response to central nervous system injury as a result of an increased proneness of females to synthesise and release PRL, which is possibly linked to the relatively more favourable prognosis of MS in women.
Assuntos
Doenças Desmielinizantes/sangue , Doenças Desmielinizantes/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Prolactina/sangue , Prolactina/líquido cefalorraquidiano , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Elevated ApoA1 levels have been associated with decreased dementia risk. The A-allele of the APOA1 -75G/A promoter polymorphism has been associated with elevated ApoA1 levels. OBJECTIVE: We sought to investigate the effect of the APOA1 -75G/A promoter polymorphism on cognitive performance in patients with multiple sclerosis (MS). METHODS: A total of 138 patients with MS and 43 controls were studied and underwent neuropsychological assessment with Rao's Brief Repeatable Battery and the Stroop test. All patients were genotyped for APOA1. RESULTS: APOA1 A-allele carriers displayed superior overall cognitive performance compared with non-carriers (P 0.008) and had a three-fold decrease in the relative risk of overall cognitive impairment (OR 0.29, 95% CI 0.11-0.74). Regarding performance on individual cognitive domains, although APOA1 A-allele carriers performed better than non-carriers on all tests, this was significant only for semantic verbal fluency and the Stroop interference task (P 0.036 and 0.018, respectively). CONCLUSIONS: We found an association of the APOA1 -75G/A promoter polymorphism with cognitive performance in MS. This effect was most prominent on semantic verbal fluency and the Stroop interference task.
Assuntos
Apolipoproteína A-I/genética , Transtornos Cognitivos/genética , Cognição , Esclerose Múltipla/genética , Polimorfismo Genético , Adulto , Transtornos Cognitivos/epidemiologia , Feminino , Predisposição Genética para Doença/epidemiologia , Genótipo , Grécia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/epidemiologia , Testes Neuropsicológicos , Regiões Promotoras Genéticas/genética , Fatores de Risco , Aprendizagem VerbalRESUMO
BACKGROUND: Spastic paretic hemifacial contracture (SPHC) is an uncommon condition, originally described as a sign of brainstem neoplasia, characterized by sustained unilateral contraction of the facial muscles associated with mild ipsilateral facial paresis. SPHC has only rarely been reported in the context of multiple sclerosis (MS). To further study and assess the frequency of SPHC in patients with MS. METHODS: We screened clinically 500 consecutive patients with MS for the presence of SPHC and further studied electrophysiologically any cases identified. RESULTS: We identified two patients who developed the condition during the course of an MS relapse. The estimated frequency of the condition was 0.4%. Both patients had relapsing-remitting MS. SPHC was characterized on Electromyography (EMG) by continuous resting activity of irregularly firing motor unit potentials, associated with impaired recruitment of motor units on voluntary contraction. Myokymic discharges were not present. Blink reflex studies were partly consistent with midpontine lesions in the vicinity of the facial nucleus ipsilateral to SPHC. MRI showed lesions in the ipsilateral dorsolateral midpontine tegmentum. CONCLUSIONS: SPHC constitutes a rare but distinct clinical and EMG entity in patients with MS.
Assuntos
Espasmo Hemifacial/diagnóstico , Esclerose Múltipla Recidivante-Remitente/diagnóstico , Espasticidade Muscular/diagnóstico , Paresia/diagnóstico , Adulto , Piscadela , Eletromiografia , Fácies , Feminino , Espasmo Hemifacial/etiologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Esclerose Múltipla Recidivante-Remitente/complicações , Espasticidade Muscular/etiologia , Condução Nervosa , Paresia/etiologiaRESUMO
OBJECTIVE: To investigate the effect of APOE epsilon4 on different cognitive domains in a population of Greek patients with multiple sclerosis (MS). METHODS: A total of 125 patients with MS and 43 controls were included in this study and underwent neuropsychological assessment with Rao's Brief Repeatable Battery. All patients with MS were genotyped for APOE. The effect of APOE epsilon4 on different cognitive domains was investigated. RESULTS: Fifty-one percent of patients with MS were cognitively impaired. E4 carriers had a sixfold increase in the relative risk of impairment in verbal learning vs noncarriers (OR 6.28, 95% CI 1.74 to 22.69). This effect was domain-specific and was not observed in other cognitive domains assessed by the battery. CONCLUSION: We found an association of APOE epsilon4 with impaired verbal learning in patients with multiple sclerosis.
Assuntos
Apolipoproteína E4/genética , Transtornos Cognitivos/genética , Predisposição Genética para Doença/genética , Deficiências da Aprendizagem/genética , Esclerose Múltipla/genética , Adulto , Transtornos Cognitivos/epidemiologia , Transtornos Cognitivos/psicologia , Comorbidade , Análise Mutacional de DNA , Avaliação da Deficiência , Feminino , Marcadores Genéticos/genética , Testes Genéticos , Variação Genética/genética , Genótipo , Grécia/epidemiologia , Humanos , Deficiências da Aprendizagem/epidemiologia , Deficiências da Aprendizagem/psicologia , Masculino , Esclerose Múltipla/epidemiologia , Esclerose Múltipla/psicologia , Testes Neuropsicológicos , Fatores de Risco , Comportamento Verbal/fisiologiaRESUMO
Rana temporaria sartorius muscle fibres were exposed to varied sequences of solution and temperature changes that have been employed hitherto in procedures that sought to decouple the transverse tubules from the surface membrane. The incidence of such detubulation was assessed in large numbers of fibres through demonstrating a loss or otherwise of the after-depolarization that normally reflects successful tubular propagation of the surface action potential. This criterion yielded assessments of the existing detubulation techniques in agreement with earlier results. The experiments then developed an improved detubulation procedure that required only brief (15 min) exposures to glycerol, its replacement in a single step by a Ca2+/Mg(2+)-Ringer solution for 30 min, and rapid cooling from room temperature (19-21 degrees C) to 6-10 degrees C prior to final restoration of the normal Ringer solution. This sequence of steps yielded an optimal incidence (98%) of detubulation in viable surface fibres that were amenable to electrophysiological studies. Studies that systematically modified the detubulation procedure demonstrated that the omission of any one step in the protocol significantly reduced the incidence of detubulation with or without accompanying deteriorations in fibre resting potentials. Successful detubulation accordingly required an initial exposure to an optimal glycerol concentration that lasted for a minimal duration and for its abrupt withdrawal. Inclusion of a cooling step within 30 min after glycerol withdrawal was coincident with, and critical to, optimal tubular isolation. Thus, cooling steps that either preceded, or that followed the glycerol withdrawal step by more than 60 min, resulted in a sharp reduction in the incidence of detubulation. Similarly, a critical period of exposure to Ca2+/Mg2+ Ringer solution also promoted detubulation without compromising the recovery of stable and satisfactory resting potentials. The findings reported here remain consistent with a primarily osmotic mechanism for detubulation. However, they demonstrated additional and important influences of temperature and of divalent cation concentration on the extent of tubular detachment when such factors were modified during the time course of the expected volume changes that followed each adjustment in osmotic condition.