Dual-modal Assessment for in Vivo Investigation of Neurovascular Characteristic of Cerebral Edema Induced by Lipopolysaccharide

: The pathological features of cerebral edema are complicated, but usually only intracranial pressure (ICP) is regarded as the most important indicator for monitoring cerebral edema. The research focused on investigating the neurovascular characteristic of the lipopolysaccharide (LPS)-induced cerebral edema model in rats by using simultaneous electrophysical and hemodynamic recording. The results showed that neurophysiology (firing rate (FR), interval histogram index (ISI), and the power spectrum of local field potential (LFPs power)) and hemodynamic response (oxygenated hemoglobin (HbO 2 ), deoxyhemoglobin (HbR) and relative cerebral blood flow (CBF)) were linearly related, and the Pearson’s correlation coefficient was determined by the BBB integrity after LPS injection . Furtherly, the improvement of treatment after two agents were observed successfully through these neurophysiological and hemodynamic parameters. The optical-electrical joint method provided a technical solution for cerebral edema functional monitoring and anti-edema drug efficacy evaluation. Our findings revealed the neurovascular and BBB impact of cerebral edema and improved the limitation of in vivo pathological diagnosis of cerebral edema. therapeutic response in a rat model using simultaneous electrophysical and hemodynamic recording. Comprehensive neurovascular and BBB information, including the FR, ISI, LFPs power, CBF, HbO 2 , and HbR, could be acquired and assessed concurrently to analyze neurovascular dynamic changes exhibited by the LPS-induced cerebral edema model. Our results suggested that cerebral edema may spontaneously cause blood flow disorder and neural activity abnormity due to the damage of BBB. The increased ICP by brain edema induced by LPS restricted neuronal activity, which subsequently attenuated metabolic responses and accelerated cerebral blood flow. We believe that the assessment method proposed in the present paper could provide a feasible technical solution for clinical edema pathological monitoring and drug efficacy evaluation.

the impairment of neurovascular function was found during brain disorders including cerebral edema. For example, Matilde et al. found that subarachnoid hemorrhage could result in acute changes in the cerebral microcirculation 4 . The monitoring of neuronal activity, blood oxygen and cerebral blood flow is thus necessary and helpful for understanding the fundamental pathophysiology of edema development as well as for identifying possible therapeutic targets of cerebral edema. For example, Brad et al. monitored that brain activity and the time-dependent neurophysiological and hemodynamic response during the cerebral ischaemia and reperfusion process 5 .
One of the key mechanisms underlying the formation of cerebral edema is the disruption of the blood brain barrier (BBB), which plays an essential role in maintaining the balance of water-electrolyte distribution inside and outside the neurons and glia 6 .
In the meantime, a large body of evidence has indicated that BBB is the core structure for modulating the brain circulation and neurovascular function 7 . Nevertheless, few studies have investigated the pathological characteristics of cerebral edema from the perspective of the neural activity 8 . Therefore, we herein aim to elucidate the possible relationship between neuronal and hemodynamic parameters so as to bridge the complexity of BBB during the progression of cerebral edema. In addition, the use of LPS in the experiment was taken into consideration that LPS could activate the expression of inflammatory factors, such as IL-Iβ, TNF-α, et al., the factors could initiate or contribute to the BBB injury and eventually cause the formation of cerebral edema 9 .
The aim of this paper is to systematically explore the neural activity and hemodynamic response during the progression of LPS induced by cerebral edema and to evaluate the effects of anti-edema drugs. The optical-electrical joint system was developed to record electrophysiological and hemodynamic parameters concurrently and the assessment method can feedback the neurovascular and BBB characteristic of cerebral edema from the term of neural activity and hemodynamic response, which enabled providing a potential technical solution for clinical cerebral edema pathological development monitoring and drug efficacy evaluation.

Animal preparation
All in vivo animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Nanjing University of Aeronautics and Astronautics. The research was approved by institutional animal care and Use committee of Nanjing Medical university (IACUC-1909023). Female Sprague Dawley rats (140-180 g) from Animal Experiment Center of Nanjing Medical University (Nanjing, China) were housed in cages with food and water ad libitum. All rats were maintained in a room with a 12h light/dark cycle and allowed a 3-day adaptation prior to experiments. The rats were randomly assigned to 6 groups, approximately 10 in each group and the experiment procedure for each group is listed in Table.1. The rats in G1 and G2 groups were injected intravenously with LPS and saline respectively at the beginning of the experiment. Afterwards, the data were recorded every 20 minutes till 140 min after the injection. The rats in the G3, G4, and G5 groups were injected with mannitol (MA), hypertonic saline (HS), and saline respectively at 120 min after the LPS injection, and afterwards the corresponding data was recorded every 20 min till 220 min. The rats injected with saline during the entire process (G6) were used as control. In the experimental recording process, the rats were kept under a moderately anesthetized state, and the heart rate was at approximately 350 bpm.

Surgical procedures
Surgical procedures were performed after animals were deeply anesthetized with 10% pentobarbital (165 mg/kg). The animal anesthesia and operation procedures were described previously in details 10 . Briefly, the cranial hole was roughly 1.

Dual-modal System setup and recording procedures
The schematic representation of our multimodal system is shown in Fig.1 for recording electrophysical and hemodynamic parameters, including spikes, CBF, HbO2 and HbR.
Briefly, the integrated optical-electrical monitoring platform is composed of the neuroelectrophysiology recording system, the intrinsic optical signal (IOS) spectrum acquisition system, and the laser speckle imaging platform. For avoiding the influence of laser on electrophysiological signal collection, the laser speckle contrast images were obtained immediately after the electrophysiological signals were recorded for 5 minutes.

Spikes and local field potentials (LFPs) recording
The rats were implanted with a 2 × 2 microelectrode array (diameter about 33 µm, Here, n t is the firing time of the nth spike, and is the firing time of the nth +1 spike.
Then the mean ISI was calculated during 0.2s epoch after stimulation and regarded as an indicator of spike coding pattern.

Blood oxygen saturation measurement
Broadband spectrum was recorded from the in-house built spectrum measurement apparatus. A dual-fiber probe consisted of two optical fibers with the diameter of 200 μm and core-to-core distance of 200 μm was used to collect the optical signals. The excitation light with the wavelength from 200 ~ 1100 nm was coupled into one of the optical fibers. Then the light scattered back from the samples was collected by the other optical fiber and was detected by a spectrometer (USB 2000, Ocean Optics, USA). The obtained raw spectrum data were converted into changes of HbO2 and HbR by the IOS analysis, which was carried out by the modified Beer-Lambert law 12 and least squares, seen in Eq.2.
10 0 where ε is the molar absorptivity, is the path length factor, ΔC is the change in molar concentration compared to baseline measurement, a I is the post light intensity, and 0 I is the baseline light intensity. Molar absorptivity and path length factor values were obtained from previous studies reported by Kuboyama et al. 13 . The concentrations of both HbO2 and HbR can be distinguished by applying least squares to the following relationship, seen in Eq.3.
Here,  to scattering coefficient and s  was the relative change of chromophore scattering.
The following six wavelengths of light 450nm, 470nm, 500nm, 550nm, 570 nm and 600 nm were selected in consideration of the appropriate absorption peaks and equivalent absorption points of HbO2, HbR, Cytaa3-D, Cytc-D and FAD, which can be regarded as the temporal response for HbO2 and HbR.

Brain blood flow observation using laser speckle contrast imaging
Laser speckle contrast imaging was employed to visualize the changes of brain blood flow during the entire experimental stages. In details, a He-Ne laser was adjusted by using a collimator (632.8 nm and 15 mW, Thorlabs, USA). Then the laser passed an expander and irradiated the rat brain region with a diameter of ~12 mm with an incident angle of 30°-45°. The illuminated region was magnified through a zoom stereo microscope (50486A, Navitar, USA), and the raw speckle images were captured through a monochrome CCD camera (12 bit, Point Grey, Canada) with 2448×2048 pixels. The exposure time of the CCD was set as 20 ms, and the images were acquired through the programming software at 20 Hz. Then, a stack of 30 raw images were stored and processed by using Matlab (Mathworks, MA). The following equation Eq.4 was used to calculate speckle contrast ( K ).
where  was the standard deviation and I  was the mean of intensities within a sliding window. Here, a 5×5 pixels spatial window was used as the convolution window.
The CBF was calculated in Eq.5 with the above sliding window, and the detailed algorithm was followed by our previous study and introduced earlier 14 .
The speckle contrast value at each pixel of the captured image was calculated to obtain the velocity distribution map, showing the pattern of blood flow on the cortical surface. For quantitative evaluation, regions of interest (ROIs) of CBF were selected in LSCI images and the CBF had a running average of 1 s before analysis of changes in response to LSP injection as baseline.

Brain water content measurement
At the end of the experiment, all rats were sacrificed by decapitation under the deep anesthesia state, and the brains were removed immediately. The cortical tissues (about 1 mm 3 in volume) was extracted and placed on the electronic analytical balance to measure the wet weight (WW). The dry weight (DW) was measured after the brain tissue was dried for 24 h above 100 °C. The percent of brain water content was obtained from the Eq.6 Water content (%) = (WW − DW) × 100%/WW (6)

Immunohistochemistry analysis
In this study, IL-1β, TNF-α and IgG were used to evaluate the pathological characteristics of cerebral edema by ELISA kit. The detailed experimental operation was introduced earlier literature 15 . The Optical Density (O.D.) at 450 nm could be distinguish using a spectrometer.

Statistics analysis
Statistical analysis was performed using SPSS software (SPSS Statistics v. 19.0, IBM Corp, Armonk, USA) by one sample t test, one-way analysis of variance (ANOVA) test and repeated measures ANOVA followed by Dunnet's post-hoc test. Differences were considered to be statistically significant at p < 0.05 and p<0.01. For correlation analysis, Pearson correlation coefficient was determined between two variables. p < 0.05 was considered statistically significant.

RESULTS
In Fig  In consideration of the physiological difference between different rats, the calculated CBF and blood oxygen were normalized, and averaged data in the control group before any injection was used for normalization. As shown in Fig.3 (a) Similarly, the spikes and LFPs data were also normalized and analyzed. In Fig.3   Taken together, the above results indicated that hemodynamic responses to LPS might appear earlier than the neuronal response in terms of electrical signal changes. To further identify the underlying mechanism of the therapeutic effects of MA and HS on LPS-induced cerebral edema, cortical tissues in each group were collected and stained with Evans Blue dye, which can only extravasate into leaked blood-brain barrier. Fig.5 (a) showed Evans Blue staining images of rat brains in each group. Obviously, Evans Blue yielded a deeper penetration in G5 of which rats did not receive any therapeutic intervention, and no obvious difference was found between the G3, G4 group, Besides, the faliure of Evans Blue staining infiltration in the G6 group indicated BBB integrity.
Furthermore, the ICP value, the water content of the brain tissues and the concentrations of multiple inflammatory mediators including IL-1β, TNF-α and IgG in different groups were analyzed shown in Fig.5 (b-f). The G5 group rendered the highest ICP, water content and the highest concentrations of all tested inflammatory factors as compared to other groups, which indicated that LPS indeed induced the accumulation of a large amount of water and inflammatory mediators. In addition, the ICP, the water content and the concentrations of inflammatory mediators in the G3 and G4 treatment groups were significantly lower than those in the G5 group, yet higher than the normal condition, which demonstrated the consistency of the optical-electrical joint system in evaluating the therapeutic effects of MA and HS from the biochemical side.

DISCUSSION
The purpose of the paper is to analyze the electrophysiological signals and blood oxygen signals collected by the developed optical-electrical joint system, to study the relationship of neural activity and hemodynamic response during the progression of LPS-induced cerebral edema, and to evaluate pharmaceutical efficacy anti-edema drugs through the correlation response. Numbers of studies have proved that LPS stimulated a variety of inflammatory factors, such as, IL-Iβ, TNF-α, IgG, caused BBB damage, and eventually contributed to cerebral edema. But the pathological progression of cerebral edema induced by LPS was complex 26 , it is difficult to accurately monitor the pathological changes of LPS only by relying on ICP measurement, MRI or inflammatory factor immunoassay methods.
In the paper, we continuously recorded the electrophysical parameters (FR, and LFPs power), and the hemodynamic parameters (CBF, HbO2, HbR), which could capture neurovascular function with microscopic vascular and electrophysiological resolution.
Our results showed that, hemodynamic and neuronal activity could reflect a physiologically complemental progress of LPS-induced by cerebral edema.
In the initial stage immediately after LPS injection, all of the monitored parameters have not demonstrated significant changes until 40 min in the LPS-treated group in comparison with the control group. Yet afterwards, FR, ISI, LFPs power and HbO2 started to decrease while HbR and CBF began to rise markedly from 40 min to 120 min. The time-dependent response to LPS has thus proved the correlation between the neural activity of the brain and the local cerebral blood flow. Here we inferred LPSinduced cerebral edema was highly likely to be due to changes in cerebral blood flow and blood oxygen caused by permeability destruction of the BBB.
During the late stage of LPS action, LPS might reach the brain tissue through the disruptive BBB and regulate the expression of AQP-4 in glial cells, which is dominating the process of active transport of water molecules 27 and resulted in the rapid accumulation of a large amount of water and abnormal neural activity. Indeed, the expression of inflammatory mediators in the cortex associated with progressive BBB disruption was identified, and the result is consistent with hemodynamic and electrophysiological response.
Afterwards, two therapeutic agents, MA and HS solution, were then injected into the rats through the tail vein 28 . The results demonstrated that hemodynamic and electrophysiological parameters gradually recovered and the therapeutic agent indeed relieved the condition of brain edema. Futhermore, the comparison of Pearson's correlation coefficient between FR and hemodynamic parameters pre-or post-antiedema drug administration (time point: 120 min) suggested that neurovascular characteristics was based on edema pathology. Thrane found that the AQP-4 increased significantly after about 100 minutes, and AQP-4 was positively correlated with cytotoxic brain edema 29 . Similarly, the Pearson's correlation within 140-220 min declined by more than 50% compared to that within 0-120 min in Fig.6(b). In the further research of the determination coefficient R 2 , by analyzing R 2 of the 0-120 min and 140-220 min in G5 group, the results indicated the confidence and satisfaction of the Pearson correlation between hemodynamic and neuronal activity completely changed.
The pathological characteristics of LPS induced cerebral edema transformed from simple BBB permeability to water retention inside and outside nerve cells 30 . Due to BBB is the key to maintain the normal working environment of neurons and glial cells.
by identifying the integrity of the BBB function, the neurovascular characteristic parameters are used as the basis for the evaluation of the pathological progression of cerebral edema.

CONCLUSIONS
an optical-electrical joint system was developed to study the neurovascular characteristic during the progression of cerebral edema and corresponding therapeutic response in a rat model using simultaneous electrophysical and hemodynamic recording. Comprehensive neurovascular and BBB information, including the FR, ISI, LFPs power, CBF, HbO2, and HbR, could be acquired and assessed concurrently to analyze neurovascular dynamic changes exhibited by the LPS-induced cerebral edema model. Our results suggested that cerebral edema may spontaneously cause blood flow disorder and neural activity abnormity due to the damage of BBB. The increased ICP by brain edema induced by LPS restricted neuronal activity, which subsequently attenuated metabolic responses and accelerated cerebral blood flow. We believe that the assessment method proposed in the present paper could provide a feasible technical solution for clinical edema pathological monitoring and drug efficacy evaluation.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. All data generated or analysed during this study are included in this published article

Authors' contributions
The experiments were conceived and designed by Weitao Li, Yameng Zhang.
Experiments were carried out by Yameng Zhang, Qian Xie and Ning Xue. Experiments data were analyzed by Yameng Zhang and Liuye Yao. The paper was written by Yameng Zhang, Yamin Yang and edited by all authors.