研究报告: 大鼠感觉运动系统静息态脑网络研究

为了理解啮齿类动物的脑功能连接,本文利用9.4T fMRI获得轻度麻醉状态下大鼠静息状态及刺激激活的数据,通过互相关分析构建节点之间的相关系数矩阵并计算相应的网络参数．结果发现：给予前爪电刺激时,刺激对侧初级感觉皮层(S1)、丘脑(Tha)有较强的正激活,双侧尾状壳核(CPu)有较强的负激活．静息状态时大鼠感觉/运动皮层内部、丘脑内部的连接性较强,而感觉/运动皮层与丘脑之间的连接较弱,双侧感觉运动系统之间存在较强的同步低频振荡,感觉运动系统在静息态时的脑网络具有小世界属性．结果提示,啮齿类动物在大脑信息处理中的功能分离和整合可能与人类存在某些相似性,支持哺乳动物中枢神经系统的基本功能存在遗传保守性的观点．     

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.     

Functional Connectivity Estimated from Resting-State fMRI Reveals Selective Alterations in Male Adolescents with Pure Conduct Disorder

Conduct disorder (CD) is characterized by a persistent pattern of antisocial behavior and aggression in childhood and adolescence. Previous task-based and resting-state functional magnetic resonance imaging (fMRI) studies have revealed widespread brain regional abnormalities in adolescents with CD. However, whether the resting-state networks (RSNs) are altered in adolescents with CD remains unknown. In this study, resting-state fMRI data were first acquired from eighteen male adolescents with pure CD and eighteen age- and gender-matched typically developing (TD) individuals. Independent component analysis (ICA) was implemented to extract nine representative RSNs, and the generated RSNs were then compared to show the differences between the CD and TD groups. Interestingly, it was observed from the brain mapping results that compared with the TD group, the CD group manifested decreased functional connectivity in four representative RSNs: the anterior default mode network (left middle frontal gyrus), which is considered to be correlated with impaired social cognition, the somatosensory network (bilateral supplementary motor area and right postcentral gyrus), the lateral visual network (left superior occipital gyrus), and the medial visual network (right fusiform, left lingual gyrus and right calcarine), which are expected to be relevant to the perceptual systems responsible for perceptual dysfunction in male adolescents with CD. Importantly, the novel findings suggested that male adolescents with pure CD were identified to have dysfunctions in both low-level perceptual networks (the somatosensory network and visual network) and a high-order cognitive network (the default mode network). Revealing the changes in the functional connectivity of these RSNs enhances our understanding of the neural mechanisms underlying the modulation of emotion and social cognition and the regulation of perception in adolescents with CD.     

Spinal Cord Injury and Migraine Headache: A Population-Based Study

Migraine headaches are a common neurological condition, negatively impacting health and quality of life. Among potential risk factors for migraine headache, risk of migraine headaches was elevated in individuals with spinal cord injury (SCI). The association between migraines and SCI is intriguing to consider from the perspective that migraine headaches may be acquired in response to damage in the spinal cord. The primary objective of this study was to further examine the association between SCI and migraine headache, controlling for potential confounding variables. A secondary objective was to determine the impact of migraine headaches on self-perceived health. Data from a sample of 61,047 participants were obtained from the cross-sectional Canadian Community Health Survey. Multivariable logistic regression was used to explore the association between SCI and migraine headache using probability weights and adjusting for confounders. The multivariable age- and sex-adjusted model revealed a strong association between SCI and migraine headache, with an adjusted odds ratio for migraine of 4.82 (95% confidence interval [3.02, 7.67]) among those with SCI compared to those without SCI. Further, individuals who experienced both SCI and migraine tended to report poorer perceived general health compared with the other groups (i.e., SCI and no migraine). In conclusion, this study established a strong association between SCI and migraine headache. Further research is needed to explore the possible mechanisms underlying this relationship. Improvements in clinical practice to minimize this issue could result in significant improvements in quality of life.     

Changes of Functional and Effective Connectivity in Smoking Replenishment on Deprived Heavy Smokers: A Resting-State fMRI Study

Previous researches have explored the changes of functional connectivity caused by smoking with the aid of fMRI. This study considers not only functional connectivity but also effective connectivity regarding both brain networks and brain regions by using a novel analysis framework that combines independent component analysis (ICA) and Granger causality analysis (GCA). We conducted a resting-state fMRI experiment in which twenty-one heavy smokers were scanned in two sessions of different conditions: smoking abstinence followed by smoking satiety. In our framework, group ICA was firstly adopted to obtain the spatial patterns of the default-mode network (DMN), executive-control network (ECN), and salience network (SN). Their associated time courses were analyzed using GCA, showing that the effective connectivity from SN to DMN was reduced and that from ECN/DMN to SN was enhanced after smoking replenishment. A paired t-test on ICA spatial patterns revealed functional connectivity variation in regions such as the insula, parahippocampus, precuneus, anterior cingulate cortex, supplementary motor area, and ventromedial/dorsolateral prefrontal cortex. These regions were later selected as the regions of interest (ROIs), and their effective connectivity was investigated subsequently using GCA. In smoking abstinence, the insula showed the increased effective connectivity with the other ROIs; while in smoking satiety, the parahippocampus had the enhanced inter-area effective connectivity. These results demonstrate our hypothesis that for deprived heavy smokers, smoking replenishment takes effect on both functional and effective connectivity. Moreover, our analysis framework could be applied in a range of neuroscience studies.     

A Functionally Relevant Tool for the Body following Spinal Cord Injury

A tool such as a prosthetic device that extends or restores movement may become part of the identity of the person to whom it belongs. For example, some individuals with spinal cord injury (SCI) adapt their body and action representation to incorporate their wheelchairs. However, it remains unclear whether the bodily assimilation of a relevant external tool develops as a consequence of altered sensory and motor inputs from the body or of prolonged confinement sitting or lying in the wheelchair. To explore such relationships, we used a principal component analysis (PCA) on collected structured reports detailing introspective experiences of wheelchair use in 55 wheelchair-bound individuals with SCI. Among all patients, the regular use of a wheelchair induced the perception that the body’s edges are not fixed, but are instead plastic and flexible to include the wheelchair. The PCA revealed the presence of three major components. In particular, the functional aspect of the sense of embodiment concerning the wheelchair appeared to be modulated by disconnected body segments. Neither an effect of time since injury nor an effect of exposure to/experience of was detected. Patients with lesions in the lower spinal cord and with loss of movement and sensation in the legs but who retained upper body movement showed a higher degree of functional embodiment than those with lesions in the upper spinal cord and impairment in the entire body. In essence, the tool did not become an extension of the immobile limbs; rather, it became an actual tangible substitution of the functionality of the affected body part. These findings suggest that the brain can incorporate relevant artificial tools into the body schema via the natural process of continuously updating bodily signals. The ability to embody new essential objects extends the potentiality of physically impaired persons and can be used for their rehabilitation.     

Decreased Prefrontal Lobe Interhemispheric Functional Connectivity in Adolescents with Internet Gaming Disorder: A Primary Study Using Resting-State fMRI

Purposes

Recent neuroimaging studies have shown that people with Internet gaming disorder (IGD) have structural and functional abnormalities in specific brain areas and connections. However, little is known about the alterations of the interhemispheric resting-state functional connectivity (rsFC) in participants with IGD. In the present study, we used a newly developed voxel-mirrored homotopic connectivity (VMHC) method to investigate the interhemispheric rsFC of the whole brain in participants with IGD.

Methods

We compared interhemispheric rsFC between 17 participants with IGD and 24 healthy controls, group-matched on age, gender, and education status. All participants were provided written informed consent. Resting-state functional and structural magnetic resonance images were acquired for all participants. The rsFC between bilateral homotopic voxels was calculated. Regions showing abnormal VMHC in IGD participants were adopted as regions of interest for correlation analyses.

Results

Compared to healthy controls, IGD participants showed decreased VMHC between the left and right superior frontal gyrus (orbital part), inferior frontal gyrus (orbital part), middle frontal gyrus and superior frontal gyrus. Further analyses showed Chen Internet Addiction Scale (CIAS)-related VMHC in superior frontal gyrus (orbital part) and CIAS (r = −0.55, p = 0.02, uncorrected).

Conclusions

Our findings implicate the important role of altered interhemispheric rsFC in the bilateral prefrontal lobe in the neuropathological mechanism of IGD, and provide further supportive evidence for the reclassification of IGD as a behavioral addiction.     

Alterations in Low-Level Perceptual Networks Related to Clinical Severity in PTSD after an Earthquake: A Resting-State fMRI Study

Background

Several task-based functional MRI (fMRI) studies have highlighted abnormal activation in specific regions involving the low-level perceptual (auditory, visual, and somato-motor) network in posttraumatic stress disorder (PTSD) patients. However, little is known about whether the functional connectivity of the low-level perceptual and higher-order cognitive (attention, central-execution, and default-mode) networks change in medication-naïve PTSD patients during the resting state.

Methods

We investigated the resting state networks (RSNs) using independent component analysis (ICA) in 18 chronic Wenchuan earthquake-related PTSD patients versus 20 healthy survivors (HSs).

Results

Compared to the HSs, PTSD patients displayed both increased and decreased functional connectivity within the salience network (SN), central executive network (CEN), default mode network (DMN), somato-motor network (SMN), auditory network (AN), and visual network (VN). Furthermore, strengthened connectivity involving the inferior temporal gyrus (ITG) and supplementary motor area (SMA) was negatively correlated with clinical severity in PTSD patients.

Limitations

Given the absence of a healthy control group that never experienced the earthquake, our results cannot be used to compare alterations between the PTSD patients, physically healthy trauma survivors, and healthy controls. In addition, the breathing and heart rates were not monitored in our small sample size of subjects. In future studies, specific task paradigms should be used to reveal perceptual impairments.

Conclusions

These findings suggest that PTSD patients have widespread deficits in both the low-level perceptual and higher-order cognitive networks. Decreased connectivity within the low-level perceptual networks was related to clinical symptoms, which may be associated with traumatic reminders causing attentional bias to negative emotion in response to threatening stimuli and resulting in emotional dysregulation.     

Altered Spontaneous Brain Activity in Patients with Acute Spinal Cord Injury Revealed by Resting-State Functional MRI

Background

Previous neuroimaging studies have provided evidence of structural and functional reorganization of brain in patients with chronic spinal cord injury (SCI). However, it remains unknown whether the spontaneous brain activity changes in acute SCI. In this study, we investigated intrinsic brain activity in acute SCI patients using a regional homogeneity (ReHo) analysis based on resting-state functional magnetic resonance imaging.

Methods

A total of 15 patients with acute SCI and 16 healthy controls participated in the study. The ReHo value was used to evaluate spontaneous brain activity, and voxel-wise comparisons of ReHo were performed to identify brain regions with altered spontaneous brain activity between groups. We also assessed the associations between ReHo and the clinical scores in brain regions showing changed spontaneous brain activity.

Results

Compared with the controls, the acute SCI patients showed decreased ReHo in the bilateral primary motor cortex/primary somatosensory cortex, bilateral supplementary motor area/dorsal lateral prefrontal cortex, right inferior frontal gyrus, bilateral dorsal anterior cingulate cortex and bilateral caudate; and increased ReHo in bilateral precuneus, the left inferior parietal lobe, the left brainstem/hippocampus, the left cingulate motor area, bilateral insula, bilateral thalamus and bilateral cerebellum. The average ReHo values of the left thalamus and right insula were negatively correlated with the international standards for the neurological classification of spinal cord injury motor scores.

Conclusion

Our findings indicate that acute distant neuronal damage has an immediate impact on spontaneous brain activity. In acute SCI patients, the ReHo was prominently altered in brain regions involved in motor execution and cognitive control, default mode network, and which are associated with sensorimotor compensatory reorganization. Abnormal ReHo values in the left thalamus and right insula could serve as potential biomarkers for assessment of neuronal damage and the prediction of clinical outcomes in acute SCI.     

Respiratory Nursing Interventions Following Tracheostomy in Acute Traumatic Cervical Spinal Cord Injury

Tracheostomy is frequently performed in severe cervical spinal cord injury (SCI) patients with the pulmonary dysfunction. A series of respiratory nursing interventions are required to plan tracheostomy removal. Tracheostomy was performed in 29 patients after acute traumatic cervical SCI. A series of respiratory nursing interventions were introduced in these patients after closed tracheostomy and decannulation, including closed tracheostomy tube training, manually assisted cough. Chacheostomy was successfully removed in 21 patients after the respiratory nursing interventions. In contrast, eight patients died from associated injuries. The average time from tracheostomy to decannulation was 40 days (14–104 days), the average time from closed tracheostomy to decannulation was 18.80 ± 13.50 days. Second tracheostomy was performed in one patient after 29 days’ removal due to pulmonary infection. One patient presented with delayed incision healing for 29 days. Closed tracheostomy tube training and manually assisted cough are key factors for tracheostomy removal, although intensive nursing are also needed. The time from tracheostomy to decannulation and from closed tracheostomy to decannulation is increased in case of "late" (>24 h) tracheostomy and longer mechanical ventilation.     

Multimodal fMRI Resting-State Functional Connectivity in Granulin Mutations: The Case of Fronto-Parietal Dementia

Background

Monogenic dementias represent a great opportunity to trace disease progression from preclinical to symptomatic stages. Frontotemporal Dementia related to Granulin (GRN) mutations presents a specific framework of brain damage, involving fronto-temporal regions and long inter-hemispheric white matter bundles. Multimodal resting-state functional MRI (rs-fMRI) is a promising tool to carefully describe disease signature from the earliest disease phase.

Objective

To define local connectivity alterations in GRN related pathology moving from the presymptomatic (asymptomatic GRN mutation carriers) to the clinical phase of the disease (GRN- related Frontotemporal Dementia).

Methods

Thirty-one GRN Thr272fs mutation carriers (14 patients with Frontotemporal Dementia and 17 asymptomatic carriers) and 38 healthy controls were recruited. Local connectivity measures (Regional Homogeneity (ReHo), Fractional Amplitude of Low Frequency Fluctuation (fALFF) and Degree Centrality (DC)) were computed, considering age and gender as nuisance variables as well as the influence of voxel-level gray matter atrophy.

Results

Asymptomatic GRN carriers had selective reduced ReHo in the left parietal region and increased ReHo in frontal regions compared to healthy controls. Considering Frontotemporal Dementia patients, all measures (ReHo, fALFF and DC) were reduced in inferior parietal, frontal lobes and posterior cingulate cortex. Considering GRN mutation carriers, an inverse correlation with age in the posterior cingulate cortex, inferior parietal lobule and orbitofrontal cortex was found.

Conclusions

GRN pathology is characterized by functional brain network alterations even decades before the clinical onset; they involve the parietal region primarily and then spread to the anterior regions of the brain, supporting the concept of molecular nexopathies.     

Decreased Interhemispheric Coordination in Treatment-Resistant Depression: A Resting-State fMRI Study

Background

Previous studies have demonstrated that patients with treatment-resistant depression (TRD) and treatment-sensitive depression (TSD) differed at neural level. However, it remains unclear if these two subtypes of depression differ in the interhemispheric coordination. This study was undertaken for two purposes: (1) to explore the differences in interhemispheric coordination between these two subtypes by using the voxel-mirrored homotopic connectivity (VMHC) method; and (2) to determine if the difference of interhemispheric coordination can be used as a biomarker(s) to differentiate TRD from both TSD and healthy subjects (HS).

Methods

Twenty-three patients with TRD, 22 with TSD, and 19 HS participated in the study. Data of these participants were analyzed with the VMHC and seed-based functional connectivity (FC) approaches.

Results

Compared to the TSD group, the TRD group showed significantly lower VMHC values in the calcarine cortex, fusiform gyrus, hippocampus, superior temporal gyrus, middle cingulum, and precentral gyrus. Lower VMHC values were also observed in the TRD group in the calcarine cortex relative to the HS group. However, the TSD group had no significant change in VMHC value in any brain region compared to the HS group. Receiver operating characteristic curves (ROC) analysis revealed that the VMHC values in the calcarine cortex had discriminatory function distinguishing patients with TRD from patients with TSD as well as those participants in the HS group.

Conclusions

Lower VMHC values of patients with TRD relative to those with TSD and those in the HS group in the calcarine cortex appeared to be a unique feature for patients with TRD and it may be used as an imaging biomarker to separate patients with TRD from those with TSD or HS.     

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.¹ Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5° (animals rotated at 22.5°), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya''s staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.     

扩散张量成像对颈椎病脊髓早期损伤的研究

目的:探讨磁共振(MR)扩散张量成像(DTI)作为定量分析方法,对脊髓型颈椎病(CSM)脊髓早期损伤诊断的应用价值.方法:选择45例经临床及影像诊断为脊髓型颈椎病患者,颈椎常规MRI检查显示脊髓内无异常信号,使用单次激发自旋回波平面(SE-EPI)序列,进行DTI扫描.测量压迫部位脊髓的ADC值及FA值作为病例组,选择病变上或下方两个节段以上未受压正常脊髓作为正常对照组,测量其ADC值及FA值.分析病例组与对照组间ADC及FA值差别,计算ADC值及FA值诊断脊髓损伤的敏感性.结果:所有脊髓型颈椎病患者经DTI检查均可得到ADC图及FA图,经图像后处理,脊髓显示清晰,图像无变形及伪影.3例脊髓型颈椎病患者ADC值降低,42例脊髓型颈椎病患者ADC值增高,平均ADC值为(1.388± 0.149)x 10-3 mm2/s.44名脊髓型颈椎病患者FA值降低,1名脊髓型颈椎病患者FA值增高,平均FA值为0.476±0.085,受压处脊髓平均ADC值升高,平均FA值下降,与正常值比较差别有统计学意义.ADC值诊断的敏感性为93.33％,FA值诊断的敏感性为97.78％.结论:DTI与常规MR比较,能早期而准确地诊断脊髓型颈椎病脊髓早期损伤.     

Altered Fronto-Striatal and Fronto-Cerebellar Circuits in Heroin-Dependent Individuals: A Resting-State fMRI Study

Background

The formation of compulsive pattern of drug use is related to abnormal regional neural activity and functional reorganization in the heroin addicts’ brain, but the relationship between heroin-use-induced disrupted local neural activity and its functional organization pattern in resting-state is unknown.

Methodology/Principal Findings

With fMRI data acquired during resting state from 17 male heroin dependent individuals (HD) and 15 matched normal controls (NC), we analyzed the changes of amplitude of low frequency fluctuation (ALFF) in brain areas, and its relationship with history of heroin use. Then we investigated the addiction related alteration in functional connectivity of the brain regions with changed ALFF using seed-based correlation analysis. Compared with NC, the ALFF of HD was obviously decreased in the right caudate, right dorsal anterior cingulate cortex (dACC), right superior medial frontal cortex and increased in the bilateral cerebellum, left superior temporal gyrus and left superior occipital gyrus. Of the six regions, only the ALFF value of right caudate had a negative correlation with heroin use. Setting the six regions as “seeds”, we found the functional connectivity between the right caudate and dorsolateral prefrontal cortex (dlPFC) was reduced but that between the right caudate and cerebellum was enhanced. Besides, an abnormal lateral PFC-dACC connection was also observed in HD.

Conclusions

The observations of dysfunction of fronto-striatal and fronto-cerebellar circuit in HD implicate an altered balance between local neuronal assemblies activity and their integrated network organization pattern which may be involved in the process from voluntary to habitual and compulsive drug use.     

Bowel Dysfunction in Spinal Cord Injury: Current Perspectives

Permanent disruptions of gastrointestinal function are very common sequel of spinal cord injury (SCI). When motor and sensory nervous integrity are severely affected, neurogenic gastrointestinal dysfunction is an inevitable consequence. Autonomic nervous system miss function has significantly diminished or lost sensory sensations followed with incomplete evacuation of stool from the rectal vault, immobility, and reduced anal sphincter tone all of those predisposing to increased risk of fecal incontinence (FI). The FI is, beside paralysis of extremities, one of the symptoms most profoundly affecting quality of life (QOL) in patients with SCI. We are reviewing current perspectives in management of SCI, discussing some pathophysiology mechanisms which could be addressed and pointing toward actual practical concepts in use for evaluation and improvements necessary to sustain SCI patients QOL.     

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine¹. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities^2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target⁴. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI⁵. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.⁶ In comparison to other peptides, QL6 self-assembles into β-sheets at neutral pH⁶.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.