Multi-modal Optical Neurovascular Imaging In Conscious Animals:Methods And Applications

The technological advances in optical imaging,electrical engineering and computer science have ushered in a new age of neuroimaging,in which neuroimaging of neurovascular organization and cortical function has been applied in conscious and unrestrained rodents.Conventional neuroimaging technology requires animals to be restrained and anesthetized,since the crucial role of the stability of imaging in conventional neuroimaging systems with great computational and structural com-plexity,which limits the observations of cerebral blood flow（CBF）and oxygenation changes that result from neural activity in both physiological and pathological processes.The advances of optical neuroimaging in conscious and unrestrained animals shed new light on neurovascular organization,cortical function and disease mechanisms,without the interferences of anesthetics and restrains.It is in 2001,to the best of our knowledge,that the miniaturization of optical neuroimaging systems was first introduced.Recent breakthroughs in laser speckle imaging（LSI）and optical intrinsic signal imaging（OISI）,with their advantages of full-field real-time imaging in high spatiotemporal resolution,has enabled additional technological advances in the miniaturization of optical neuroimaging system.Based on LSI and OISI,innovative multi-modal optical neurovascular imaging technology is able to image the changes in CBF and oxygenation,observe the neurovascular anatomy and function,and benefit the study on disease mechanisms.In the beginning of the dissertation,we highlighted recent advances in the miniaturization of optical neurovascular imaging systems and their applications in a wide array of research fields.With the novel design of the multi-modal optical neurovascular imaging system,we introduced a single-trial estimation method of the cerebral metabolic rate of oxygen（CMRO₂）based on LSI and imaging photoplethys-mography（iPPG）.In addition,we presented a series of experiments on photothrom-botic stroke model and traumatic brain injury（TBI）consecutively,as representative applications of the multi-modal optical neurovascular imaging.The major contributions and conclusions of the dissertation are summarized as follows.（1）We proposed a novel design of multi-modal neurovascular imaging system.OISI and LSI have been used for years in the study of CBF,oxygenation,and hemodynamic responses to the neural activity under functional stimulation.In the dissertation,we designed a miniature head-mounted multi-modal neurovascular imaging system that was able to monitor the coupling of local oxygenation and CBF in conscious and unrestrained animals in real time by integrating different imaging modalities.As an application,we utilized the system in monitoring local oxygenation and CBF in a conscious and running wheel training rat.During the running wheel training,increase in total hemoglobin concentration and CBF was observed.However,OISI and LSI images showed different trends after running wheel training,i.e.total hemo-globin concentration remained at a relatively high level with a delayed tendency of decrease,while CBF returned immediately to the similar level as that before training.The preliminary results implied that the neural activity and hemodynamic response contained both linear and nonlinear components.（2）We proposed a single-trial estimation method of CMRO₂.CMRO₂ could conventionally be measured by combining laser Doppler flow-metry and multispectral reflectance imaging across multiple trials of stimulation,which compromised the real-time capacity.Monitoring transient change of CMRO₂in conscious and unrestrained animals has been challenging.In this dissertation,iPPG and LSI were combined into the multi-modal optical neurovascular imaging system for single-trial estimation of CMRO₂ with advanced real-time capacity.In a physio-logically stable experiment,the iPPG-based method showed a less than 4%variance in comparison with the conventional method over 20 trials,and its temporal stability could be comparable to that by conventional method with over 6 trials.While the oxygen supply was decreased deliberately,the new method was able to detect the transient changes of CMRO₂ in real time,which could hardly be revealed by the conventional method.（3）We proposed a series of representative applications of the multi-modal optical neurovascular imaging in conscious and unrestrained animals.In this dissertation,a series of representative applications were presented in aspect of experimental stroke and TBI studies of conscious and unrestrained animals.In experimental stroke research,anesthesia is common and serves as a major reason for translational failure.In addition,real-time CBF monitoring during stroke onset can provide important information for the prediction of the stroke outcome;however,this is difficult to achieve in clinical practice due to various technical problems.To address this,we created a photothrombotic focal ischemic stroke model in conscious and unrestrained rats utilizing the multi-modal optical neurovascular imaging system.In this model,a high spatiotemporal resolution imaging sensor using LSI was integrated to acquire real-time two-dimensional CBF information during thrombosis.The feasibility,stability,and reliability of the model were tested in terms of CBF,behavior,and T2-weighted magnetic resonance imaging（MRI）findings.After completion of occlusion,CBF in the targeted cortex was reduced to 16±9%of the baseline value acquired before modeling.The averaged infarct volume measured by MRI 24 h post modeling was 77±11 mm³ and correlated well with CBF reduction area（R²=0.74）.This rodent model of focal cerebral ischemia and real-time blood flow imaging opens the possibility of performing various fundamental and trans-lational studies on stroke without the influence of anesthetics.In another study,we utilized the photothrombotic focal ischemic stroke model in conscious and unrestrained rats,and investigated transient hemodynamic changes with or without isoflurane administration.Significantly larger CBF reduction area was observed in conscious rats from 8 minutes immediately after the onset of stroke modeling,compared with anesthetized rats.Stroke rats without isoflurane admin-istration also showed larger lesion volume identified by MRI 3 hours post occlusion（57.0%）,higher neurological severity score 24 hours post occlusion（28.3%）,and larger infarct volume from 2,3,5-triphenyltetrazolium chloride staining 24 hours post occlusion（46.9%）.Our results demonstrated that the hemodynamic features were affected by anesthetics at as early as during the stroke induction.TBI is the leading cause of death and disability among children worldwide.Affected children often suffer from emotional,cognitive and neurological impair-ments throughout life.In the controlled cortical impact animal model of pediatric TBI（postnatal day 16–17）,it was demonstrated that injury resulted in abnormal neuronal hypoactivity in the non-injured primary somatosensory cortex（S1）.It materializes that reshaping the abnormal post-injury neuronal activity may provide a suitable strategy to augment rehabilitation.We tested whether high-frequency,non-invasive transcranial magnetic stimulation（TMS）delivered twice a week over a four-week period could rescue the neuronal activity and improve the long-term functional neuro-physiological and behavioral outcome.The results showed that TBI rats subjected to TMS therapy showed significant increases in the evoked-fMRI cortical responses（189%）,evoked synaptic activity（46%）,evoked neuronal firing（200%）and expression of cellular markers of neuroplasticity in the non-injured S1 compared to TBI rats that did not receive therapy.The multi-modal optical neurovascular imaging was further applied showing complex changes in changes in CBF and oxygenation,while the conscious and unrestrained animals were exploring in the open field.In summary,we presented the novel design of the multi-modal optical neuro-vascular imaging system based on OISI and LSI.The new imaging methods,systems and representative applications show great potentials in the investigations of brain function and brain injury.