Bioresorbable silicon electronics for transient spatiotemporal mapping of electrical activity from the cerebral cortex.

Bioresorbable silicon electronics technology offers unprecedented opportunities to deploy advanced implantable monitoring systems that eliminate risks, cost and discomfort associated with surgical extraction. Applications include postoperative monitoring and transient physiologic recording after percutaneous or minimally invasive placement of vascular, cardiac, orthopaedic, neural or other devices. We present an embodiment of these materials in both passive and actively addressed arrays of bioresorbable silicon electrodes with multiplexing capabilities, which record in vivo electrophysiological signals from the cortical surface and the subgaleal space. The devices detect normal physiologic and epileptiform activity, both in acute and chronic recordings. Comparative studies show sensor performance comparable to standard clinical systems and reduced tissue reactivity relative to conventional clinical electrocorticography (ECoG) electrodes. This technology offers general applicability in neural interfaces, with additional potential utility in treatment of disorders where transient monitoring and modulation of physiologic function, implant integrity and tissue recovery or regeneration are required.

Clinical studies and anti-inflammatory mechanisms of treatments.

In this exciting era, we are coming closer and closer to bringing an anti-inflammatory therapy to the clinic for the purpose of seizure prevention, modification, and/or suppression. At present, it is unclear what this approach might entail, and what form it will take. Irrespective of the therapy that ultimately reaches the clinic, there will be some commonalities with regard to clinical trials. A number of animal models have now been used to identify inflammation as a major underlying mechanism of both chronic seizures and the epileptogenic process. These models have demonstrated that specific anti-inflammatory treatments can be effective at both suppressing chronic seizures and interfering with the process of epileptogenesis. Some of these have already been evaluated in early phase clinical trials. It can be expected that there will soon be more clinical trials of both "conventional, broad spectrum" anti-inflammatory agents and novel new approaches to utilizing specific anti-inflammatory therapies with drugs or other therapeutic interventions. A summary of some of those approaches appears below, as well as a discussion of the issues facing clinical trials in this new domain.

Issues related to development of new antiseizure treatments.

This report represents a summary of the discussions led by the antiseizure treatment working group of the International League Against Epilepsy (ILAE)/American Epilepsy Society (AES) Working Groups joint meeting in London (London Meeting). We review here what is currently known about the pharmacologic characteristics of current models of refractory seizures, both for adult and pediatric epilepsy. In addition, we address how the National Institute of Neurological Disorders and Stroke (NINDS)-funded Anticonvulsant Screening Program (ASP) is evolving to incorporate appropriate animal models in the search for molecules that might be sufficiently novel to warrant further pharmacologic development. We also briefly address what we believe is necessary, going forward, to achieve the goal of stopping seizures in all patients, with a call to arms for funding agencies, the pharmaceutical industry, and basic researchers.

Expanding applications of deep brain stimulation: a potential therapeutic role in obesity and addiction management.

BACKGROUND: The indications for deep brain stimulation (DBS) are expanding, and the feasibility and efficacy of this surgical procedure in various neurologic and neuropsychiatric disorders continue to be tested. This review attempts to provide background and rationale for applying this therapeutic option to obesity and addiction. We review neural targets currently under clinical investigation for DBS­the hypothalamus and nucleus accumbens­in conditions such as cluster headache and obsessive-compulsive disorder. These brain regions have also been strongly implicated in obesity and addiction. These disorders are frequently refractory, with very high rates of weight regain or relapse, respectively, despite the best available treatments. METHODS: We performed a structured literature review of the animal studies of DBS, which revealed attenuation of food intake, increased metabolism, or decreased drug seeking. We also review the available radiologic evidence in humans, implicating the hypothalamus and nucleus in obesity and addiction. RESULTS: The available evidence of the promise of DBS in these conditions combined with significant medical need, support pursuing pilot studies and clinical trials of DBS in order to decrease the risk of dietary and drug relapse. CONCLUSIONS: Well-designed pilot studies and clinical trials enrolling carefully selected patients with obesity or addiction should be initiated.

Antiepileptogenesis and disease modification: Clinical and regulatory issues.

This is a summary report of clinical and regulatory issues discussed at the 2018 NINDS workshop, entitled "Accelerating Therapies for Antiepileptogenesis and Disease Modification." The intent of the workshop was to optimize and accelerate development of therapies for antiepileptogenesis (AEG) and disease modification in the epilepsies. The working group discussed nomenclature for antiepileptogenic therapies, subdividing them into "antiepileptogenic therapies" and "disease modifying therapies," both of which are urgently needed. We use the example of traumatic brain injury to explain issues and complexities in designing a trial for disease-preventing antiepileptogenic therapies, including identifying timing of intervention, selecting the appropriate dose, and the need for biomarkers. We discuss the recent trials of vigabatrin to prevent onset and modify epilepsy outcome in children with tuberous sclerosis (Epistop and PreVeNT). We describe a potential approach to a disease modification trial in adults, using patients with temporal lobe epilepsy. Finally, we discuss regulatory hurdles for antiepileptogenesis and disease-modifying trials.

Posttraumatic epilepsy: the challenge of translating discoveries in the laboratory to pathways to a cure.

Translating laboratory discoveries into successful therapies for preventing epilepsy is a difficult task, but preventing epilepsy in those who are known to be at high risk needs to be one of our highest priorities. At present, we need to approach this task as a parallel set of research endeavors-one concentrating on laboratory experiments designed to learn how to prevent epilepsy after brain trauma and the other focusing on how to perform the appropriate clinical research in humans to demonstrate that whatever is discovered in the laboratory can be appropriately tested. It is too important to let the second process await conclusion of the first. Initially, we need to create a consortium of groups in trauma centers that are dedicated to antiepileptogenic studies and develop funding sources for long-term studies. We need to experiment with clinical protocols, making the studies as cost-effective as possible, while performing continuous data mining of outcomes and surrogate markers. The limitations of current technology to assist in antiepileptogenesis trials must be acknowledged: There is no currently available method for continuously monitoring electroencephalography (EEG) over prolonged periods, and there are no validated biomarkers for the process of epileptogenesis. As we learn more about the process of epileptogenesis and its underlying mechanisms, it is hoped that we will be able to prevent the development of epilepsy after traumatic brain injury (TBI) and after many other known epileptogenic lesions.

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity.

Most current methods of gene delivery for primary cultured hippocampal neurons are limited by toxicity, transient expression, the use of immature neurons and/or low efficiency. We performed a direct comparison of seven serotypes of adeno-associated virus (AAV) vectors for genetic manipulation of primary cultured neurons in vitro. Serotypes 1, 2, 7, 8 and 9 mediated highly efficient, nontoxic, stable long-term gene expression in cultured cortical and hippocampal neurons aged 0-4 weeks in vitro; serotypes 5 and 6 were associated with toxicity at high doses. AAV1 transduced over 90% of all cells with approximately 80% of the transduced cells being neurons. The method was readily adapted to a high-throughput format to demonstrate neurotrophin-mediated neuroprotection from glutamate toxicity in cultured neurons at 2 weeks in vitro. These vectors should prove highly useful for efficient overexpression or downregulation of genes in primary neuronal cultures at any developmental stage.

Human immunodeficiency virus (HIV)-induced neurotoxicity: roles for the NMDA receptor subtypes.

Neuronal damage in human immunodeficiency virus type 1 (HIV-1) infection in the brain is thought to occur at least in part through NMDA receptor (NMDAR) excitation initiated by soluble neurotoxins from HIV-infected brain macrophages. Furthermore, brain regions enriched in NMDAR-2A (NR2A) and NMDAR-2B (NR2B) subunits, such as the hippocampus, are particularly vulnerable. Using cultured rat hippocampal cells and HIV-1-infected human monocyte-derived macrophages (HIV/MDM), we examined the role of NR2A and NR2B in HIV/MDM-induced hippocampal neuronal death. We used the primary HIV-1 strain Jago derived from the CSF of an individual with HIV-associated dementia and that robustly replicates in MDM. We found the following: (1) hippocampal neuronal susceptibility to HIV/MDM excitotoxins varies according to the developmental expression patterns of NR2A and NR2B; (2) NMDAR activation by HIV/MDM results in neuronal calpain activation, which results in neuronal death; and (3) selective antagonists of homomeric NR2B/NR2B- and heteromeric NR2A/NR2B-containing NMDARs, as well as an inhibitor of calpain activity, afford neuroprotection against HIV/MDM. These studies establish a clear link between macrophage HIV infection, neuronal NR2A and NR2B activation, and calpain-mediated hippocampal neuronal death. They further suggest a dominant role for NR2A and NR2B in determining neuronal susceptibility in HIV-infected brain. Antagonists of NR2A and NR2B subunits as well as inhibitors of calpain activation offer attractive neuroprotective approaches against HIV in both developing and mature brain.

The novel antiepileptic agent RWJ-333369-A, but not its analog RWJ-333369, reduces regional cerebral edema without affecting neurobehavioral outcome or cell death following experimental traumatic brain injury.

PURPOSE: To evaluate the therapeutic efficacy of two antiepileptic compounds, RWJ-333369 and RWJ-333369-A in a well-established experimental model of lateral fluid percussion (FP) traumatic brain injury (TBI) in the rat. METHODS: Anethestized Male Sprague-Dawley rats (n=227) were subjected to lateral FP brain injury or sham-injury. Animals were randomized to receive treatment with RWJ-333369 (60 mg/kg, p.o.) or its analog RWJ-333369-A (60 mg/kg, p.o.), or vehicle (equal volume) at 15 minutes, 4, 8, and 24 hours post-injury. In Study I, animals were assessed at 48 hours for acute motor and cognitive function and then sacrificed to evaluate regional cerebral edema. In Study II, animals were evaluated post-injury for motor function at 48 hours and weekly thereafter from 1 to 4 weeks. Post-traumatic learning ability was assessed 4 weeks post-injury, followed by evaluation of hemispheric tissue loss. RESULTS: In Study I, no improvement in acute memory or motor function was observed following administration of either RWJ-333369 or RWJ-333369-A in brain-injured animals compared to vehicle-treated, brain-injured animals. However, brain-injured animals receiving treatment with RWJ-333369-A had a significant reduction in post-traumatic cerebral edema in both injured and contralateral hippocampus compared to brain-injured, vehicle-treated controls (p<0.05). In Study II, treatment with either compound did not result in any improvement of neuromotor function, learning ability or change in lesion volume following brain injury. CONCLUSION: These results indicate that the novel antiepileptic compound RWJ-333369-A reduces post-traumatic hippocampal edema without affecting neurobehavioral or histological outcome. It remains unclear whether this small effect on hippocampal edema ie related to the ability of this compound to attenuate seizure activity.

Effects of rapamycin on gene expression, morphology, and electrophysiological properties of rat hippocampal neurons.

PURPOSE: We assayed the effects of rapamycin, an immunomodulatory agent known to inhibit the activity of the mammalian target of rapamycin (mTOR) cascade, on candidate gene expression and single unit firing properties in cultured rat hippocampal neurons as a strategy to define the effects of rapamycin on neuronal gene transcription and excitability. METHODS: Rapamycin was added (100nM) to cultured hippocampal neurons on days 3 and 14. Neuronal somatic size and dendritic length were assayed by immunohistochemistry and digital imaging. Radiolabeled mRNA was amplified from single hippocampal pyramidal neurons and used to probe cDNA arrays containing over 100 distinct candidate genes including cytoskeletal element, growth factor, transcription factor, neurotransmitter, and ion channel genes. In addition, the effects of rapamycin (200nM) on spontaneous neuronal activity and voltage-dependent currents were assessed. RESULTS: There were no effects of rapamycin on cell size or dendrite length. Rapamycin altered expression of distinct mRNAs in each gene family on days 3 and 14 in culture. Single unit recordings from neurons exposed to rapamycin exhibited no change from baseline. When spontaneous activity was increased by blocking GABA-mediated inhibition with bicuculline, a fraction of the neurons exhibited a decreased duration of spontaneous bursts and a decrease in synaptic inputs. Rapamycin did not appear to alter voltage-dependent Na(+) or K(+) currents underlying action potentials. CONCLUSIONS: These data demonstrate that rapamycin does not produce neurotoxicity nor alter dendritic growth and complexity in vitro and does not significantly alter voltage-gated sodium and potassium currents. Rapamycin does affect neuronal gene transcription in vitro. Use of rapamycin in clinical trials for patients with tuberous sclerosis complex warrants vigilance for possible effects on seizure frequency and neurocognitive function.

Murine brain progenitor cells have the ability to differentiate into functional neurons and integrate into the CNS.

Although neural stem and progenitor cells have been shown to differentiate into neurons, few studies have examined the physiological properties of the differentiated neurons derived from stem cells. Here we show that mouse brain progenitor cells (mBPCs) differentiated in culture by removal of mitogenic factors or addition of BDNF or GDNF express neuronal-specific proteins including MAP-2 and synaptobrevin II. However, these cells demonstrate small voltage-gated Na+ currents and are not able to generate action potentials. When the mBPCs are cocultured with developing rat hippocampal neurons, the stem cells differentiate into neurons expressing MAP-2, develop large voltage-gated Na+ currents, and are able to generate action potentials. To investigate the influence of a mature CNS environment on survival, differentiation, migration, and morphological integration, mBPCs were transplanted into the spinal cord of adult mice. Undifferentiated cells transplanted into the spinal cord exhibited limited migration and expressed NG2, but did not differentiate to express MAP-2. Predifferentiated cells migrated to both gray and white matter with about 23% cells developing MAP-2 immunoreactivity after 8 weeks. These results suggest that both the environment and state of differentiation may dictate migration and the differentiation pathway of stem cells after transplantation.

Models of epileptogenesis in adult animals available for antiepileptogenesis drug screening.

Epileptogenesis is the process by which parts of a normal brain are converted to a hyperexcitable brain, often after an injury. Researchers must understand this process before they know where and how to change it. Animal models are used to evaluate the process of epileptogenesis by studing status epelepticus, electrical kindling, or other methods that provoke injuries. All are associated with neuronal loss to more or less degree, synaptic reorganization, axon sprouting, neurogenesis, gliosis, and changes in gene expression in neurons and astrocytes. He describes several types of animal models and how they might be useful in developing effective strategies for preventing epilepsy.

Treatment-responsive limbic encephalitis identified by neuropil antibodies: MRI and PET correlates.

We report seven patients, six from a single institution, who developed subacute limbic encephalitis initially considered of uncertain aetiology. Four patients presented with symptoms of hippocampal dysfunction (i.e. severe short-term memory loss) and three with extensive limbic dysfunction (i.e. confusion, seizures and suspected psychosis). Brain MRI and [(18)F]fluorodeoxyglucose (FDG)-PET complemented each other but did not overlap in 50% of the patients. Combining both tests, all patients had temporal lobe abnormalities, five with additional areas involved. In one patient, FDG hyperactivity in the brainstem that was normal on MRI correlated with central hypoventilation; in another case, hyperactivity in the cerebellum anticipated ataxia. All patients had abnormal CSF: six pleocytosis, six had increased protein concentration, and three of five examined had oligoclonal bands. A tumour was identified and removed in four patients (mediastinal teratoma, thymoma, thymic carcinoma and thyroid cancer) and not treated in one (ovarian teratoma). An immunohistochemical technique that facilitates the detection of antibodies to cell surface or synaptic proteins demonstrated that six patients had antibodies to the neuropil of hippocampus or cerebellum, and one to intraneuronal antigens. Only one of the neuropil antibodies corresponded to voltage-gated potassium channel (VGKC) antibodies; the other five (two with identical specificity) reacted with antigens concentrated in areas of high dendritic density or synaptic-enriched regions of the hippocampus or cerebellum. Preliminary characterization of these antigens indicates that they are diverse and expressed on the neuronal cell membrane and dendrites; they do not co-localize with VGKCs, but partially co-localize with spinophilin. A target autoantigen in one of the patients co-localizes with a cell surface protein involved in hippocampal dendritic development. All patients except the one with antibodies to intracellular antigens had dramatic clinical and neuroimaging responses to immunotherapy or tumour resection; two patients had neurological relapse and improved with immunotherapy. Overall, the phenotype associated with the novel neuropil antibodies includes dominant behavioural and psychiatric symptoms and seizures that often interfere with the evaluation of cognition and memory, and brain MRI or FDG-PET abnormalities less frequently restricted to the medial temporal lobes than in patients with classical paraneoplastic or VGKC antibodies. When compared with patients with VGKC antibodies, patients with these novel antibodies are more likely to have CSF inflammatory abnormalities and systemic tumours (teratoma and thymoma), and they do not develop SIADH-like hyponatraemia. Although most autoantigens await characterization, all share intense expression by the neuropil of hippocampus, with patterns of immunolabelling characteristic enough to suggest the diagnosis of these disorders and predict response to treatment.

Differential localization of ErbB receptor ensembles influences their signaling in hippocampal neurons.

Our studies indicate that ErbB complexes participate in both survival and synaptic plasticity signals of hippocampal neurons but in a manner that depends on the subcellular localization of the receptor ensembles. Using dissociated hippocampal cultures, we found that neurons, rather than glial cells, are the primary targets of ErbB receptor ligands such as epidermal growth factor and heregulin. Further investigation demonstrated that ErbB receptors distribute differentially in hippocampal neurons with the epidermal growth factor receptor confined to neural cell bodies and the p185(c-neu) and ErbB4 receptors distributed to both neural soma and neurites. Activation of ErbB receptor and downstream signaling molecules were observed in neurites only after heregulin stimulation. The receptor complex which mediated neurite located signals was the p185(c-neu)/ErbB4 heterodimer. Colocalization of p185(c-neu), but not epidermal growth factor receptor, with postsynaptic density protein 95 suggests that the heregulin signaling contributes to synapse specific activities. However, the epidermal growth factor receptor complex mediates physiological survival signals, as neuronal survival was enhanced by epidermal growth factor, rather than heregulin. Collectively, these studies indicate that different ErbB ensembles localize to different locations on the neuron to mediate distinct signals and functions.

Electrophysiologic recordings in traumatic brain injury.

Following a traumatic brain injury (TBI), the brain undergoes numerous electrophysiologic changes. The most common techniques used to evaluate these changes include electroencepalography (EEG) and evoked potentials. In animals, EEGs immediately following TBI can show either diffuse slowing or voltage attenuation, or high voltage spiking. Following a TBI, many animals display evidence of hippocampal excitability and a reduced seizure threshold. Some mice subjected to severe TBI via a fluid percussion injury will eventually develop seizures, which provides a useful potential model for studying the neurophysiology of epileptogenesis. In humans, the EEG changes associated with mild TBI are relatively subtle and may be challenging to distinguish from EEG changes seen in other conditions. Quantitative EEG (QEEG) may enhance the ability to detect post-traumatic electrophysiologic changes following a mild TBI. Some types of evoked potential (EP) and event related potential (ERP) can also be used to detect post-traumatic changes following a mild TBI. Continuous EEG monitoring (cEEG) following moderate and severe TBI is useful in detecting the presence of seizures and status epilepticus acutely following an injury, although some seizures may only be detectable using intracranial monitoring. CEEG can also be helpful for assessing prognosis after moderate or severe TBI. EPs, particularly somatosensory evoked potentials, can also be useful in assessing prognosis following severe TBI. The role for newer technologies such as magnetoencephalography and bispectral analysis (BIS) in the evaluation of patients with TBI remains unclear.

High copy wildtype human 1N4R tau expression promotes early pathological tauopathy accompanied by cognitive deficits without progressive neurofibrillary degeneration.

INTRODUCTION: Accumulation of insoluble conformationally altered hyperphosphorylated tau occurs as part of the pathogenic process in Alzheimer's disease (AD) and other tauopathies. In most AD subjects, wild-type (WT) tau aggregates and accumulates in neurofibrillary tangles and dystrophic neurites in the brain; however, in some familial tauopathy disorders, mutations in the gene encoding tau cause disease. RESULTS: We generated a mouse model, Tau4RTg2652, that expresses high levels of normal human tau in neurons resulting in the early stages of tau pathology. In this model, over expression of WT human tau drives pre-tangle pathology in young mice resulting in behavioral deficits. These changes occur at a relatively young age and recapitulate early pre-tangle stages of tau pathology associated with AD and mild cognitive impairment. Several features distinguish the Tau4RTg2652 model of tauopathy from previously described tau transgenic mice. Unlike other mouse models where behavioral and neuropathologic changes are induced by transgenic tau harboring MAPT mutations pathogenic for frontotemporal lobar degeneration (FTLD), the mice described here express the normal tau sequence. CONCLUSIONS: Features of Tau4RTg2652 mice distinguishing them from other established wild type tau overexpressing mice include very early phenotypic manifestations, non-progressive tau pathology, abundant pre-tangle and phosphorylated tau, sparse oligomeric tau species, undetectable fibrillar tau pathology, stability of tau transgene copy number/expression, and normal lifespan. These results suggest that Tau4RTg2652 animals may facilitate studies of tauopathy target engagement where WT tau is driving tauopathy phenotypes.

Differential effects of the anticonvulsant topiramate on neurobehavioral and histological outcomes following traumatic brain injury in rats.

The efficacy of topiramate, a novel therapeutic agent approved for the treatment of seizure disorders, was evaluated in a model of traumatic brain injury (TBI). Adult male rats were anesthetized (sodium pentobarbital, 60 mg/kg, i.p.), subjected to lateral fluid percussion brain injury (n = 60) or sham injury (n = 47) and randomized to receive either topiramate or vehicle at 30 min (30 mg/kg, i.p.), and 8, 20 and 32 h postinjury (30 mg/kg, p.o.). In Study A, memory was evaluated using a Morris water maze at 48 h postinjury, after which brain tissue was evaluated for regional cerebral edema. In Study B, animals were evaluated for motor function at 48 h and 1, 2, 3, and 4 weeks postinjury using a composite neuroscore and the rotating pole test and for learning ability at 4 weeks. Brains were analyzed for hemispheric tissue loss and hippocampal CA3 cell loss. Topiramate had no effect on posttraumatic cerebral edema or histologic damage when compared to vehicle. At 48 h, topiramate treatment improved memory function in sham but not brain-injured animals, while at one month postinjury it impaired learning performance in brain-injured but not sham animals. Topiramate significantly improved composite neuroscores at 4 weeks postinjury and rotating pole performance at 1 and 4 weeks postinjury, suggesting a potentially beneficial effect on motor function following TBI.

Adenosine Receptor Subtypes and Cardioprotection.

Brief ischemia prior to a sustained period of ischemia reduces myocardial infarct size, a phenomenon known as preconditioning. A cardiac ventricular myocyte model has been developed to investigate the role and signaling mechanism of adenosine receptor subtypes in cardiac preconditioning. A 5-min exposure of cardiac myocytes to simulated ischemia, termed preconditioning ischemia, prior to a subsequent 90-min period of ischemia protected them against injury incurred during the 90-min ischemia. Preconditioning ischemia preserved ATP content, reduced percentage of cells killed, and decreased release of creatine kinase into the medium. Activation of the adenosine A1 receptor with CCPA or the A3 receptor with IB-MECA can replace preconditioning ischemia and mimic the protective effect of preconditioning ischemia. Blockade of the A1 receptor with its selective antagonist DPCPX or of the A3 receptor with the A3 selective antagonist MRS1191 during the preconditioning ischemia resulted in only a partial attenuation of the subsequent protection. Incubation with both DPCPX and MRS1191 or with the nonselective antagonist 8-SPT during the preconditioning ischemia completely abolished the protective effect of preconditioning ischemia. The KATP channel opener pinacidil caused a large activation of the KATP channel current and was able to precondition the myocyte. The KATP channel antagonist glibenclamide blocked the cardioprotective effect of preconditioning ischemia when it was included during myocyte exposure to the preconditioning ischemia, indicating that KATP channel is a requisite effector in mediating preconditioning. A receptor-mediated stimulation of phospholipase C or phospholipase D, with consequent activation of protein kinase C and KATP channel, appears to be the signaling mechanism linking adenosine A1 and A3 receptors to the induction of preconditioning. A model of how ischemic preconditioning is triggered and mediated is proposed. Evidence is accumulating to support its validity.

Anti-epileptogenic clinical trial designs in epilepsy: issues and options.

Although trials with anti-seizure drugs have not shown anti-epileptogenic or disease-modifying activity in humans, new compounds are on the horizon that may require novel trial designs. We briefly discuss the unique challenges and the available options to identify innovative clinical trial designs that differentiate novel anti-epileptogenic and disease-modifying compounds, preferably early in phase II, from current anti-seizure drugs. The most important challenges of clinical testing of agents for epilepsy prevention include having sufficient preclinical evidence for a suitable agent to proceed with a human trial of an anti-epileptogenic drug, and to demonstrate the feasibility of doing such a trial. Major challenges in trial design to assess agents for disease modification include the choice of suitable study parameters, the identification of a high-risk study population, the type of control, the time and duration of treatment, and a feasible follow-up period.