Carbogen gas-challenge BOLD fMRI in assessment of liver hypoxia after portal microcapsules implantation.

BACKGROUND: Hypoxia is one of the key factors affecting the survival of islet cells transplanted via the portal vein. Blood oxygen level dependent functional magnetic resonance imaging (BOLD-fMRI) is the only imaging technique that can detect the level of blood oxygen level in vivo. However, so far no study has indicated that BOLD-fMRI can be applied to monitor the liver oxygen level after islet transplantation.
OBJECTIVE: To evaluate the value of Carbogen-challenge BOLD MRI in assessing the level of hypoxia in liver tissue after portal microcapsules implanted.
METHODS: Fifty-one New Zealand rabbits were randomly divided into three experimental groups (15 in each group) were transplanted microencapsulated 1000 microbeads/kg (PV1 group), 3000 microbeads/kg (PV2 group), 5000 microbeads/kg (PV3 group), and 6 rabbits were injected with the same amount of saline as the control group, BOLD-fMRI was performed following carbogen breathing in each group after transplantation on 1d, 2d, 3d and 7d, T2* weighted image, R2* value and ΔR2* value parameters for the liver tissue. Pathological examinations including liver gross pathology, H&E staining and pimonidazole immunohistochemistry were performed after BOLD-fMRI. The differences of pathological results among each group were compared. The ΔR2* values and transplanted doses were analyzed. RESULTS AND
CONCLUSIONS: ΔR2* values at the 1-3d and 7d after transplantation were significantly different in each groups (P<0.05). ΔR2* values decreased gradually with the increase of transplanted dose, and was negatively correlated with transplant dose at 3d after transplantation (r = -0.929, P <0.001). Liver histopathological examination showed that the degree of hypoxia of liver tissue increased with the increase of transplanted doses, Carbogen-challenge BOLD-fMRI can assess the degree of liver hypoxia after portal microcapsules implanted, which provided a monitoring method for early intervention.

Introduction

Islet cell transplantation is an ideal treatment of type I diabetes mellitus and can effectively prevent the progress of diabetes mellitus [1-3]. However, due to the immunological rejection after transplantation leading to impaired islet graft function, patients often have to re-take the exogenous insulin or re-transplant [4-5]. Microencapsulated pancreatic islet cells with translucent membrane provide a possibility to solve immunological rejection [6-8]. However, the long-term follow-up results are poor due to hypoxia and other problems. The rate of patients who do not rely on exogenous insulin within 5 years after transplantation is only 8.5% [9-11].

It is believed that the survival of islet cells after transplantation depends mainly on the revascularization of blood supply, which originates from the host. The vascular system of the host promotes the regeneration of blood vessels, while the endothelial cells retained when the islet cells are separated participate in the process of revascularization. However, the revascularization will not begin immediately. It usually starts about 2–4 days after the operation and takes about 10–14 days to complete. Before the establishment of blood supply, islet cells mainly depended on oxygen permeation with surrounding liver parenchyma to maintain their function and survival. In order to achieve the therapeutic effect of diabetes mellitus, a large amount of microcapsules transplantation is needed. Large dose of microcapsules will stay in the small branches of portal vein after entering the liver, which may cause vascular embolism, leading to ischemic blood oxygen in the surrounding liver tissue, necrosis and apoptosis, seriously affecting the oxygen permeation of islet cells, thereby leading to islet cell damage, and impairing the liver function, affecting the therapeutic effect [12-15]. In order to alleviate the degree of hypoxia and hepatic vascular embolism, the main method currently used is to undergo multiple rounds of transplantations. However, with the increase of transplantation times, the probability of recipient infection is rising, which also greatly increases the cost of transplantation and economic burden for those patients. Therefore, how to solve the above contradictions according to individual differences requires a better grasp of the relationship between transplantation volume and frequency, and a quantitative detection of target hypoxia after transplantation. Moreover, the detection of hypoxia in the target area after transplantation can reflect the effectiveness of immune activation in the early stage of transplantation, the recovery level of hepatic hemodynamics, and provide relevant evidence for clinical intervention.

At present, the monitoring method of liver hypoxia level in transplantation area is limited to target tissue biopsy. This invasive method not only increases the infection rate but also the pain of recipients. Therefore, a safe, non-invasive, real-time, dynamic, effective and simple method is urgently needed to monitor the hypoxia level of liver after transplantation.

Blood oxygen level-dependent functional magnetic resonance imaging (BOLD-fMRI), acting via modulation of the T2*-weighted signal by changes in the ratio of blood paramagnetic deoxyhemoglobin to diamagnetic oxyhemoglobin, is the only technique that can detect the level of blood oxygen in vivo. It is widely used in the fields of central nervous system and liver [16-19]. By observing the changes of T2* signal, we can detect the changes of blood oxygen content in local tissues and judge the degree of hypoxia and hemodynamic changes. In this study, we simulated the clinical islet transplantation process with different number of microcapsules after portal vein transplantation in rabbits, and performed BOLD-fMRI by Carbogen administration (95% O2 and 5% CO2) at different time points after operation. The purpose of this study was to explore the relationship between ΔR2* value of liver before and after stimulation and liver hypoxia level after different dosages of microcapsules transplantation, and to further discuss the BOLD-fMRI in evaluating the hypoxia level of liver tissue after portal vein microcapsule transplantation.

Materials and methods

Animals and groups

The study was approved by the Ethical Review Committees (which is equivalent to IACUC) of the Xiangya school of medicine affiliated Haikou hospital, Central South University. We can confirm that all methods were performed in accordance with the guidelines for the care and use of experimental animals developed by the Chinese Society of Laboratory Animal Science.

Forty-one adult males and ten adult females New Zealand white rabbits (6 months old with 2.0–3.1kg body weight) were used in this experiment (provided by Animal Laboratory Center of the Third Affiliated Hospital of Xiangya Medical College, Central South University): (SCXK (Hunan) 2014/0011). The animals were raised under SPF (Specific pathogen free) condition with a 12h light-dark schedule. All animals had free access to food and water. They were randomly divided into three groups: PV1, PV2 and PV3 (fifteen animals in each group). In each group, the animals were transplanted with microcapsules of 1000 microbeads/kg, 3000 microbeads/kg and 5000 microbeads/kg, respectively. Six animals were injected with the same amount of saline as control.

Experimental method

Microcapsules were prepared in 1.5% sodium alginate solution, and then dripping into CaCl2 solution to form calcium alginate microspheres. Then they were mixed with 0.05% poly L-lysine solution, 0.15% sodium alginate solution and sodium citrate solution, respectively. The diameter of the microcapsules was about 109±26μm. The morphology of microcapsules was observed by optical microscopy. 10 ml of microcapsule suspension was counted under optical microscopy.

Portal vein microcapsule transplantation

After anesthesia with 3% pentobarbital sodium (1.0ml/kg) by intravenous injection into ear margin, the animals were fixed on the operating table in supine position. A 4-5cm incision was made. The portal vein was readily detected in the liver lobe. The microcapsules were injected slowly in the portal vein for 10-15s with a total amount of 1ml. In the control group, 1 ml saline was injected into the portal vein, and the procedure was the same as the above.

After the surgery, the animals were raised individually and treated with dexamethasone and polymyxin twice a day on the incision to prevent from infection. Every day the staff of the experimental animal center made a careful examination of the animals based on the appearance and behavior. No animal died unexpectedly before the experiments.

MRI scanning

The animals were generally anesthetized with 3% sodium pentobarbital (1.0ml/kg) by intravenous injection at auricular margin. All animals were scanned by MRI (Siemens Avanto 1.5T) on the 1st, 2nd, 3rd and 7th day after operation, fasting for 12 hours before scanning. Scanning sequence included transverse T1WI, transverse and coronal fat suppression T2WI, transverse BOLD. BOLD scanning parameters are: TR 150 ms, TE 3.4–39.2 ms, 5.0 mm sacnning thickness with 2.0mm intervals, matrix 192*78 mm, FA 30 degrees, FOV 150*60 mm2.

Air and Carbogen gas were inhaled through the mask, respectively. The first BOLD scan of the liver was performed after 10 minutes of inhalation of air, and then repeat the scan after 10 minutes of Carbogen gas inhalation. The BOLD scan was performed at a flow rate of 15 ml/s.

Image analysis and post-processing

After BOLD image scanning, the original T2* image and pseudo-color image were automatically generated. Five regions of interest were selected from the left lateral lobe, left inner lobe, right outer lobe, right inner lobe and caudate lobe of five layers near the hepatic hilum, with a total size of about 25–45 voxels. The artifacts of intrahepatic blood vessels, bile ducts and chemical displacement were avoided, and the same regions of interest were ensured before and after inhalation of air and Carbogen gas. Mean R2* value (1/T2*), and then calculate ΔR2* value, ΔR2* value = R2* air-R2* Carbogen gas.

Histopathological examination

Three animals in each experimental group were sacrificed by injection of overdosed 3% sodium pentobarbital (2.0ml/kg) at the end of 1, 2, 3 and 7 days after operation. The control group (6 animals) was sacrificed at 7 days after operation. Pimonidazole, a hypoxia marker, was injected into the abdominal cavity of the animals at a dose of 60 mg/kg 2 hours before execution. The rabbit liver tissue was taken within 30 minutes after execution. Paraffin sections were stained with HE and pimonidazole immunohistochemistry. The region was the same as the area of interest scanned in BOLD-fMRI. Immunohistochemical staining criteria: brown granules in the cytoplasm were positive cells. Five ROIs were randomly selected under microscopy (100X), and 100 hepatocytes were counted in each ROI (totally 500 cells). The data were analyzed with the method proposed by Nordsmark et al. [20]. Briefly, percentage of positive cells: positive cells accounted for less than 5%, 6%-15%, 16% - 30% and more than 30% of all cells were assigned with 1, 2,3 and 4 score, respectively. Positive staining intensity: 1 score for non-staining, 2 score for light staining, 3 score for moderate and 4 score for heavy staining, respectively. Hypoxia level = Percentage of positive cells * intensity of positive staining. If the score is in 0–3, then marked as (-). If in 4–6, 7–9, 10–12 then marked as (+), (++),(+++), respectively.

Statistical analysis

All the data were analyzed with SPSS 17.0 statistical software. The correlation between theΔR2* value and the transplantation dose was analyzed by non-parametric Spearman correlation analysis, P < 0.05 was statistically significant.

Results

Microcapsules transplantation induced defect in liver

In control group, the liver surface and parenchyma were normal; in PV1 group, the color of liver surface was normal 3 days after operation, HE staining showed enlargement of hepatic sinuses around portal vein and no degeneration and necrosis of hepatocytes; in PV2 group, a few patchy white ischemic foci appeared on the liver surface, HE staining showed enlargement of hepatic sinuses around portal vein, vacuolar degeneration of hepatocytes and scattered nuclear fragmentation in PV3 group. Visceral enlargement, a large number of flaky gray-yellow necrosis foci appeared on the surface. HE staining showed that the hepatic sinuses around the portal vein were obviously enlarged, a large number of degeneration and necrosis of hepatocytes were observed, and neutrophil infiltration was detected in the interstitium (Fig 1).

Fig 1

Morphological observation and Hematoxylin-eosin staining of liver in each group on the 3rd day after transplantation.

(A) and (E), (B) and (F), (C) and (G) were pathological and HE staining results on the 3rd day after transplantation in PV1, PV2 and PV3 groups; (D) and (H) was pathological and HE staining results on the 7th day after transplantation in the control group. In PV1 group, hepatic sinusoids enlarged slightly; in PV2 group, a small amount of hepatocytes degenerated and necrotized; in PV3 group, a large number of hepatocytes degenerated and necrotized; in control group, there was no abnormal change in hepatocytes.

Hypoxia was increased as indicated by pimonidazole staining

To investigate the hypoxia level in the liver, we did pimonidazole staining, a hypoxia indicator. The pimonidazole positive cells showed brown signaling in cytoplasm (Fig 2). After quantification, we found that in PV1 group, the hypoxia level was (+) on the 1st to 3rd and 7th day after operation. In PV2 group, the hypoxia level was (+)on the 1st to 2nd day after operation, and (++) on the 3rd day and (+) on the 7th day after operation. However, in PV3 group, the hypoxia level was (+++) on all of the time points we detected, which indicated that the most severity of hypoxia. All of the control group are negative (-)(Fig 2). These results showed that the more amount of microcapsules transplanted, the more severity of hypoxia was.

Fig 2

Immunohistochemical staining of pimonidazole in each group.

In PV1 group, the hypoxia level was (+) on the 1st to 3rd and 7th day after operation. In PV2 group, the hypoxia level was (+)on the 1st to 2nd day after operation, and (++) on the 3rd day and (+) on the 7th day after operation. In PV3 group, the hypoxia level was (+++) on all of the time points.

ΔR2* is decreased with the amount of microcapsules transplanted

The BOLD signal increased after carbogen gas administration (Fig 3). The ΔR2* value in each group was shown in Table 1 and Fig 4. With the increase of portal vein microcapsule transplantation dosage, theΔR2* value decreased gradually. Within 3 days after operation, theΔR2* value decreased with the prolongation of time, and increased on the 7th day after operation. When the dosage of portal vein transplantation was 5000 microbeads/kg, theΔR2* was the smallest, indicating the strongest effect. The statistical analysis was shown in Fig 4.

Fig 3

BOLD-fMRI parametric maps.

The BOLD signal increased after carbogen gas administration. With the increase of portal vein microcapsule transplantation dosage, theΔR2* value decreased gradually.

Table 1

The ΔR2* value at different time points in each group (Hz, ±s).

Time	1d	2d	3d	7d
PV1	20.32±2.31	20.00±2.60	19.45±2.01	20.27±2.31
PV2	16.78±2.34	15.81±1.69	14.99±1.40	16.10±1.40
PV3	10.30±1.67	8.51±2.15	8.70±2.00	9.21±2.34
Sham	26.03±1.42	24.07±1.54	26.07±2.06	25.90±2.11

Fig 4

Statistical analysis of the ΔR2* value in each group.

The ΔR2* values in each group after microcapsules transplanted on the 1st, 2nd, 3rd and 7th day were shown. Unpaired Student’s two tailed t-Test were used for statistical analysis, with significance levels * for P<0.05, ** for P<0.01 and *** for P<0.001.

The correlation of the ΔR2* with the amount of transplanted microcapsules and hypoxia level

We noticed that after 3 days of transplantation, the hypoxia level increased severely, then we detected the correlation of ΔR2* with hypoxia level and the amount of transplanted microcapsules on this timepoint. On the 3rd day after transplantation, theΔR2* value decreased with the increase of transplantation dose. Whilst, we also detected the most strong signaling level with pimonidazole staining. Nonparametric Spearman test showed that theΔR2* was negatively correlated with transplantation dose (r = -0.932, P<0.001, Fig 5A) and pimonidazole signaling (r = -0.893, p<0.001, Fig 5B).

Fig 5

The correlation of the ΔR2* with the amount of transplanted microcapsules and hypoxia level.

The ΔR2* value was negatively correlated with the transplantation dose (r = -0.932, P<0.001, Fig 5A) and pimonidazole signaling (r = -0.893, p<0.001, Fig 5B). on the 3rd day after transplantation.

Discussion

Islet cell transplantation is an ideal method for the treatment of type I diabetes mellitus, which is simple and convenient with low adverse reactions. It has been widely used, but it faces severe immunological rejection after operation. In 1980, Lim and Sun initiated the transplantation of rat islet cells encapsulated in alginate-polylysine microcapsules [21]. It was a translucent membrane that allowed oxygen, electrolyte, small molecule nutrients and metabolites to pass through but keep the islet cells unaffected by the immune system, and provided an ideal method for solving the immunological rejection of islet cell transplantation.

Previous study found that vascular regeneration after islet cell transplantation is the key factor to ensure the survival and function of islets [22]. However, islet revascularization takes 10–14 days, and one of the important factors affecting islet survival is hypoxia [23-24]. In the early stage of liver microencapsulated islet transplantation, the oxygen consumed by islet cells are mainly supplied by diffusion at the transplantation site. Staying in small branches of portal vein with large doses of microcapsules may cause vascular embolism, leading to ischemia in surrounding liver tissue and then seriously affecting the survival of islet cells. Therefore, the early stage after transplantation is an important factor affecting the success or failure of transplantation. For the liver function damage caused by hypoxia after transplantation, several rounds of transplantation are often used clinically to reduce this damage, which makes it difficult to monitor the amount of transplantation. Thus, it is necessary to setup an effective in vivo monitoring method. Sakada et al. found that the most severe liver injury occurred on the second day after islet cell portal vein transplantation, meanwhile after one month of transplantation, liver damage was less severe than that at an early stage [25]. Davalli et al. found that the number of islet cells and insulin content decreased significantly on the first to third day after transplantation. Besides of instant blood mediated inflammatory reaction at the early stage of transplantation, the ischemia-hypoxia mediated injury of recipient liver tissue is an important factor leading to the survival of islet cells [26]. In this study, we monitored hepatic hypoxia on the 1st, 2nd, 3rd and 7th day after operation, and pimonidazole immunohistochemical results were used as a reference for hypoxia. Pimonidazole is a hypoxic probe that can bind to hypoxic cells and identify them with antibodies [27-29]. This study found that the degeneration and necrosis of hepatocytes were the most serious in PV3 group on the third day. When the dose of microcapsule transplantation increased gradually, the degree of liver hypoxia increased gradually. After microcapsule transplantation, the liver hypoxia became worse gradually. On the third day, it showed the most serious stage. On the seventh day, due to the strong compensatory function of the liver, hypoxia was alleviated, which was similar to the observation of Sakada et al. [25].

BOLD-fMRI uses deoxyhemoglobin as an endogenous contrast agent to observe the changes of signal to understand the local tissue oxygen saturation and tissue oxygen content, thus reflecting the changes of tissue metabolism, hemodynamics and function. R2* value is negatively correlated with tissue oxygen content. The higher R2* value, the more deoxyhemoglobin, the lower oxygen partial pressure and the lower signal on BOLD image. Previous studies have shown that the changes of hepatic hemodynamics and venous blood flow can be understood by observing the changes of BOLD signal intensity under different conditions of oxygen stimulation [30], so as to further understand the changes of hepatic vascular structure and function. Other studies have used BOLD imaging to evaluate hepatic fibrosis [17,31-32]. Compared with normal liver, the response of fibrotic liver to Carbogen stimulation decreases due to the change of hemodynamics. It shows that the R2* value decreases before and after stimulation, and with the aggravation of fibrosis, the ΔR2* value decreases. Carbogen-mediated functional magnetic resonance imaging of liver is very sensitive to changes in hepatic hemodynamics. The less portal venous blood flow, the less response to Carbogen gas stimulation. In this study, we used the classical Carbogen stimulation method, and to reduce the influence of abdominal breathing on the image and the deviation of the ΔR2* value, we applied pressure on the abdomen with sand bags and controlled the breathing gating. The results showed that BOLD-fMRI were consistent with the pathological results. TheΔR2* value in PV1, PV2 and PV3 groups decreased significantly compared with the control group. This may be due to the increase of the dose of microcapsules. Portal venous blood flow decreased gradually, and the response of liver to Carbogen stimulation decreased gradually as indicated by the ΔR2*. We also found that the ΔR2* value decreased within 1, 2 and 3 days after operation, but increased on the 7th day. This may be due to the fact that the early compensation mechanism after microcapsule transplantation can not compensate for the decrease of liver blood flow, but it can still have a buffering effect. The degree of change of hepatic parenchymal deoxyhemoglobin concentration is relatively reduced, thereby narrowing the difference between groups. In this study, we observed a significant negative correlation between the ΔR2* value and the amount of transplantation. Therefore, BOLD-fMRI stimulated by Carbogen can assess the degree of liver hypoxia after portal vein microcapsule transplantation, providing a new means for early clinical intervention and the amount of transplantation tolerated by liver.

In conclusion, BOLD-fMRI stimulated by Carbogen can evaluate the degree of liver hypoxia after portal vein microcapsule transplantation, and provide a new means for early clinical intervention and guidance of liver tolerance.

29 Aug 2019

PONE-D-19-17327

Carbogen gas-challenge BOLD fMRI in assessment of liver hypoxia after portal microcapsules implantation

PLOS ONE

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Reviewer #1: 1. Introduction: The Purpose of using Carbogen gas and the meaning of gas-challenge in the title need to be described clearly.

2. How long did it take to perform BOLD-fMRI data acquisition?

3. Results: The authors need to provide the tables to list the ΔR2*value of BOLD-fMRI in each group，and show the statistical results。

4. There are no pathological or imaging images.

5. In Table 1, if adding the P value will make the results clearer.

6. The full name of the BOLD is blood oxygen level dependent。Similar grammatical mistakes and clerical error may exist in several sentences in the manuscript，for example page 16, the first sentence“it still has buffering effect and liver parenchymal deoxyhemoglobin concentration”。Please carefully edit the language issues with the help of the native English-speaking experts.

Reviewer #2: Introduction:

1. It is too simple for the intro section。The author mentioned hypoxia or related concept in the intro section repeatedly，please identity what exactly the mechanism of hypoxia happening after Islet cell transplantation and the concept of buffering effect by artery and portal vein in liver should be discussed in the process of hypoxia

2. The mechanism of BOLD should be expounded in the intro section。

3. In“By observing the changes of T2WI signal”，should T2WI be written as T2*？

4. Please explain why you choose the Carbogen gas as the stimulant for BOLD instead of pure oxygen，especially the effect of 5% CO2

5. Please identify what ΔR2* is。

Materials and methods

1. Please identify what SPF condition is

2. Please identify the unit of transplanted microcapsules in the 1000/kg, 3000/kg and 5000/kg

3. I can not find the information in this manuscript whether the islet cell was encapsulated into the microcapsules or not，which means nothing encapsulated in the study。If the effect to liver hypoxia by microcapsules is different between islet cell encapsulated and not，then it was the major deficiency to this study，or author should prove that there is no difference in affecting liver hypoxia between islet cell encapsulated and not。We do not use the empty microcapsules for treatment after all。

4. It is better to replace saline with the solution of microcapsules（not included microcapsules） for the control group

5. I wonder why MR scan was not performed before microcapsules transplant。It is very important to get the baseline for observing changes before and after operation。Data and experiment need to be supplemented。

6. There were 51 rabbits included but only 41 were used（three for 1,2,3,7 day in three experimental groups and 6 for control group），please explain

7. “The BOLD scan was performed at a flow rate of 15 ml/s.”，it meant the flow rate in the mask？

8. The author should clarify the amount of data for each group。As my understanding to this manuscript，author used 3*5 ROIs（15）for each group to meet the sample size requirement for statistical analysis and the author should talked about the “type I error” because there were only 3 rabbits in each group after all。Inversely，If the average value from 5 ROIs was used，the date is not enough for statistical analysis at all。

9. The author should address more details about the histopathological examination ，like the sections for whole liver or partial？like how to choose the section which is used for microscopy analysis。I do not think “Five ROIs were randomly selected”is appropriate for the inhomogeneity of hypoxia which should be talked in detail。In the end，the ROIs used in BOLD analysis and histopathological analysis should be in the same/similar location which make this study more credible。

10. In the last paragraph，should R2* be written as ΔR*？

RESULTS

1. Statistical analysis between BOLD and histopathological examinations are much needed （like some quantitative indicators for avascular necrosis or the amount of capillaries，or the pimonidazole results，et al）to guarantee the value and reliability of this study。In this study，one key evidence was missed in the relationship between the amount of used microcapsules and the ΔR2*，in which the essence of hypoxia is ischemia result from obstruction of microcapsules in small vessels，hence the result from ischemia should be quantified and compared to BOLD。I only find the descriptive words for pimonidazole results but no statistical analysis。

2. In “2.1 Histopathological examination”，please indicate the time for each observation

3. Please identify the meaning of “+”for pimonidazole results

4. In “2.3：but there was significant difference between each two groups (P < 0.05).” meant significant difference between each two groups in each day？

5. In“2.4”，are all the result from 3rd day？Please address the statistical relationship between transplantation dose and ΔR2* in other time point rather than only 3rd day

Discussion

1. In the 2rd paragraph，“and pimonidazole immunohistochemical results were used as a reference for hypoxia”is improper because there was no statistical analysis between and BOLD or transplantation dose

2. In“and the response of liver to Carbogen gas stimulation decreased gradually”，please identify what the response of liver is

3. There are many times for miswriting ΔR2* as R2*，the author should check it carefully。

4. Please identify “compensatory mechanism”in detail and it is hard to understand“it still has buffering effect and liver parenchymal deoxyhemoglobin concentration”，please make it more readable

5. Please identify what change is in“The degree of change was relatively reduced”

6. It is very sloppy to make the conclusion ：“Therefore, BOLD-fMRI stimulated by Carbogen gas can assess the degree of liver hypoxia after portal vein microcapsule transplantation”due to the key evidence was missed which can be fixed by doing statistical analysis related to pimonidazole results or some quantitative indicators for avascular necrosis or the amount of capillaries

7. It is very hard to understand the last sentence “It is worthwhile……”，please rewrite it for more readability

Figure legends:

The fig4 legend should indicate the time

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Reviewer #1: No

Reviewer #2: No

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15 Oct 2019

Response to Reviewers：

Reviewer #1: 1. Introduction: The Purpose of using Carbogen gas and the meaning of gas-challenge in the title need to be described clearly.

RE：Thank you very much. We revised our manuscript according to your nice suggestion.

2. How long did it take to perform BOLD-fMRI data acquisition?

RE：Normally one scan takes less than 10min. However, the animals needs to be treated before MRI scan. Totally, the whole procedure needs around 30min for one animal.

3. Results: The authors need to provide the tables to list the ΔR2*value of BOLD-fMRI in each group，and show the statistical results。

RE：Thank you for the suggestion, we provided a new figure in our revised manuscript.

4. There are no pathological or imaging images.

RE：Yes, we had those images, they were shown in figure 1, Thanks.

5. In Table 1, if adding the P value will make the results clearer.

RE：Thank you, as you suggested in the second question, we provide a new figure with statistical results.

6. The full name of the BOLD is blood oxygen level dependent。Similar grammatical mistakes and clerical error may exist in several sentences in the manuscript，for example page 16, the first sentence“it still has buffering effect and liver parenchymal deoxyhemoglobin concentration”。Please carefully edit the language issues with the help of the native English-speaking experts.

RE：Thank you very much. We modified the manuscript carefully and corrected some typos and mistakes.

Reviewer #2: Introduction:

1. It is too simple for the intro section. The author mentioned hypoxia or related concept in the intro section repeatedly，please identity what exactly the mechanism of hypoxia happening after Islet cell transplantation and the concept of buffering effect by artery and portal vein in liver should be discussed in the process of hypoxia

2. The mechanism of BOLD should be expounded in the intro section。

RE: for 1 and 2: Thank you so much, we modified our introduction and discussion. The questions raised by the reviewer had been addressed.

3. In“By observing the changes of T2WI signal”，should T2WI be written as T2*？

RE： Thank you for your suggestion, we corrected T2WI as T2*.

4. Please explain why you choose the Carbogen gas as the stimulant for BOLD instead of pure oxygen，especially the effect of 5% CO2

RE：Thanks for your question. Carbogen (95% O2 and 5% CO2) is widely used in experimental and clinical studies. Carbogen administration is known to be safe in humans and has been clinically applied during radiation therapy in order to raise the oxygen tension and increase the radio-sensitivity of the anoxic region. Compared with using pure oxygen, incorporating 5% CO2 is believed to counteract any oxygen-induced vasoconstriction.

5. Please identify what ΔR2* is。

RE: Thanks for your question. ΔR2* value = R2* air-R2* Carbogen gas, which was present in “1.4 Image Analysis and Post-processing”.

Materials and methods

1. Please identify what SPF condition is

RE: The SPF is specific pathogen free, we added the full name in the new manuscript.

2. Please identify the unit of transplanted microcapsules in the 1000/kg, 3000/kg and 5000/kg

RE: Thank you. The unit of microcapsules is the absolute number detected under the stereotype microscope. We added a paragraph of describe this in the method section. Thanks again.

3. I can not find the information in this manuscript whether the islet cell was encapsulated into the microcapsules or not，which means nothing encapsulated in the study。If the effect to liver hypoxia by microcapsules is different between islet cell encapsulated and not，then it was the major deficiency to this study，or author should prove that there is no difference in affecting liver hypoxia between islet cell encapsulated and not。We do not use the empty microcapsules for treatment after all。

RE: Thanks for your nice comments. Yes, you are right. We encapsulated nothing in the experiment.

In this study, what we want to provide is a convenient way to detect the oxygen level when microcapsules were used in the treatment for diabetes. No matter whether the islet cell were encapsulated, the hypoxia always happened. However, relationship of the hypoxia level and the number of microcapsules transplanted was not well studied. So in the study, we detected their correlation and also tested that BOLD-fMRI is an easy and sensitive way to estimate the level of hypoxia.

4. It is better to replace saline with the solution of microcapsules（not included microcapsules） for the control group

RE: Thank you for your suggestion. Actually, we still believe saline is a better control group, because we wash and resuspend the microcapsules in saline when it was implanted.

5. I wonder why MR scan was not performed before microcapsules transplant。It is very important to get the baseline for observing changes before and after operation。Data and experiment need to be supplemented。

RE: Thank you very much, in our experiments, in each MRI scan we had a control group as the baseline which is reasonable and quite widely used by this kind of studies.

6. There were 51 rabbits included but only 41 were used（three for 1,2,3,7 day in three experimental groups and 6 for control group），please explain

RE: Thank you for this nice question. Yes, at the beginning we prepared 15 rabbits for each group in case of unexpected dying during the surgery or after surgery. However, we were so lucky, those animals were in good conditions during and after the surgeries. Therefore, on the basis of “3R rules” (Reduction, Replacement and Refinement), we did not sacrifice all of the animals.

7. “The BOLD scan was performed at a flow rate of 15 ml/s.”，it meant the flow rate in the mask？

RE: Yes, 15ml/s means the flow rate of air and Carbogen in the mask.

8. The author should clarify the amount of data for each group。As my understanding to this manuscript，author used 3*5 ROIs（15）for each group to meet the sample size requirement for statistical analysis and the author should talked about the “type I error” because there were only 3 rabbits in each group after all。Inversely，If the average value from 5 ROIs was used，the date is not enough for statistical analysis at all。

RE: Thank you for your question. Yes, in this study we used 3 animals to meet the sample size. In the analysis of images, we chose the same region (ROI) before and after carbongen-challenge. Five ROIs were selected in each animal to calculate the ΔR2* value. In this system, 3 animals is sufficient to undergo statistical analysis.

9. The author should address more details about the histopathological examination ，like the sections for whole liver or partial？like how to choose the section which is used for microscopy analysis。I do not think “Five ROIs were randomly selected”is appropriate for the inhomogeneity of hypoxia which should be talked in detail。In the end，the ROIs used in BOLD analysis and histopathological analysis should be in the same/similar location which make this study more credible。

RE: Thanks for the nice suggestion. Yes, we used the same location for the histopathological examination with BOLD analysis, which was added in the new manuscript. Then, five ROIs were randomly selected in the location. Thanks again for your suggestion.

10. In the last paragraph，should R2* be written as ΔR*？

RE: Thank you very much. Yes, it is ΔR*, we are sorry for the carelessness.

RESULTS

1. Statistical analysis between BOLD and histopathological examinations are much needed （like some quantitative indicators for avascular necrosis or the amount of capillaries，or the pimonidazole results，et al）to guarantee the value and reliability of this study。In this study，one key evidence was missed in the relationship between the amount of used microcapsules and the ΔR2*，in which the essence of hypoxia is ischemia result from obstruction of microcapsules in small vessels，hence the result from ischemia should be quantified and compared to BOLD。I only find the descriptive words for pimonidazole results but no statistical analysis。

RE：Thank you for the wonder suggestions. A new figure was incorporated in the new manuscript, which showed the rΔR2* value in different groups at distinct timepoint. Now the results were much clearer. we can notice a negative corrlation between the amount of microcapsules transplanted and the ΔR2* value. Additionally, In this figure, the statistical data were also incorporated.

With regards to the histopathological examinations, honestly, we did not observe apparent obstruction of microcapsules in the vessels, which is also not easy to identify in clinical.

However, with the Pimondazole staining we can observe apparent difference with different amount of microcapsules and the severity was increased with time passed by. For the statistical analysis, we calculated the hypoxia level of each ROI based on the method used by Nordsmark, The difference was so dramatic. Additionally, we also plotted a new figure to show the correlation between ΔR2* and hypoxia level (Fig. 5B). As the degree of hypoxia in the liver gradually increased, the average value of ΔR2* decreased. Using the nonparametric Spearman test, the average value of ΔR2* was highly negatively correlated with the hypoxic marker, and the correlation coefficient was (r=-0.893, p<0.001).

2. In “2.1 Histopathological examination”，please indicate the time for each observation

RE: Thanks a lot, we added the timepoint for each observation.

3. Please identify the meaning of “+”for pimonidazole results

RE: Thanks for your question, we added a paragraph to describe how to calculate the results based on the widely used method, and the meaning of “-“, “+”, “++”, “+++”.

4. In “2.3：but there was significant difference between each two groups (P < 0.05).” meant significant difference between each two groups in each day？

RE: Yes, it is. To make the point clear, we plotted a new figure 4 to show the statistic result.

5. In“2.4”，are all the result from 3rd day？Please address the statistical relationship between transplantation dose and ΔR2* in other time point rather than only 3rd day

RE: Thanks a lot for this suggestion. A new figure 4 was incorporated in the new manuscript, we can notice a clear correlation of transplantation dose and ΔR2*.

Discussion

1. In the 2rd paragraph，“and pimonidazole immunohistochemical results were used as a reference for hypoxia”is improper because there was no statistical analysis between and BOLD or transplantation dose.

RE: Thanks for your nice suggestions. According your questions, we added and replaced several figures, we believe those new data can support our conclusion.

2. In“and the response of liver to Carbogen gas stimulation decreased gradually”，please identify what the response of liver is

RE: Thank you very much, the responds was indicated by ΔR2*, we modified it in the manuscript.

3. There are many times for miswriting ΔR2* as R2*，the author should check it carefully。

RE: Thank you. We are so sorry for the carelessness.

4. Please identify “compensatory mechanism”in detail and it is hard to understand“it still has buffering effect and liver parenchymal deoxyhemoglobin concentration”，please make it more readable

RE: Thanks, we modified it and made it more readable.

5. Please identify what change is in “The degree of change was relatively reduced”

RE: Thank you. We correct our manuscript carefully.

6. It is very sloppy to make the conclusion ：“Therefore, BOLD-fMRI stimulated by Carbogen gas can assess the degree of liver hypoxia after portal vein microcapsule transplantation”due to the key evidence was missed which can be fixed by doing statistical analysis related to pimonidazole results or some quantitative indicators for avascular necrosis or the amount of capillaries

RE: Thank you. According your nice suggestions, we modified our manuscript and provided the statistical analysis results and some new data.

7. It is very hard to understand the last sentence “It is worthwhile……”，please rewrite it for more readability

RE: we modified the last paragraph to make it readable.

Figure legends:

The fig4 legend should indicate the time

RE: Thank you. We corrected it.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

11 Nov 2019

Carbogen gas-challenge BOLD fMRI in assessment of liver hypoxia after portal microcapsules implantation

PONE-D-19-17327R1

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18 Nov 2019

PONE-D-19-17327R1

Carbogen gas-challenge BOLD fMRI in assessment of liver hypoxia after portal microcapsules implantation

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