Review Reports

Round 1

Reviewer 1 Report

Manuscript Number:

Mass Spectrometry Imaging (MSI) delineates thymus-centric metabolism in vivo as an effect of systemic administration of dexamethasone

Yudai Tsuji 1 ; Shinichi Yamaguchi 2 ; Tomoyuki Nakamura 3 ; Masaya Ikegawa

Till: 2021.10.29

The scope of work proposed by Yudai Tsuji  et all. focus on studies of cortex of the thymus which shall enable to predict ongoing immunosuppression by in vivo DEX-administration.

The work can be valuable for clinicians involved in autoimmune disease treatment as its tends to clarify how pathological thymus react in patients under steroid-treatment. The Authors prove the concept that apoptotic cells release certain metabolites as ‘good-bye’ signals to actively modulate outcomes in tissues.

I recommend material for publication with minor improvements pointed below.

 

Remark no 1:

Page 4, lines 152

“While 2,3-BPG marks vascular structure due to its relationship with red blood cells.”

Can You explain a little more the differences visible on Fig. 3 for 2,3-bisphosphoglycerates (2,3-BPG) for DEX treated and non-treated mice.

 

Remark no 2:

Page 8, line 226

Figure 4. MSI derived from purine and pyrimidine metabolites.

The scale of intensity on the numbers is unreadable.

 

Remark no 3:

Page 9, line 274-277

Figure 5. Visualization of energy charge (EC) status of multiple thoracic organs with or without DEX administration. … EC in the thymic cortex was extremely reduced after DEX administration. In the medulla of DEX-treated thymus, heterogeneous EC distribution was noticeable.

 

The intensity scale in the color maps is not the same for the control sample and the Dex sample, hence the comparison is difficult.

EC in the thymic cortex of DEX DEX-sample is heterogeneously distributed. Still there are areas with high intensity. Rewrite this sentence.

 

Remark no 4:

Page 4, line 180

Distribution of IMP in both control and DEX treated mice were shown in Supplementary Figure.6.

What does it tell us? Lack of description of the figure.

 

Remark no 5:

Page 4, line 193

… in blue colored compartment in Figure.5

Do You refer to Fig. 6?

 

Remark no 6:

Page 5, line 198

Figure.4

Do You refer to Fig. 4 here (Figure 4. MSI derived from purine and pyrimidine metabolites)?

Author Response

 

We really appreciate valuable comments to improve our article.

We have revised our manuscript as follows:

 

For Reviewer1

 

Remark no 1:

Page 4, lines 152

“While 2,3-BPG marks vascular structure due to its relationship with red blood cells.”

Can You explain a little more the differences visible on Fig. 3 for 2,3-bisphosphoglycerates (2,3-BPG) for DEX treated and non-treated mice.

 

We appreciate this suggestion and add the following description in the text;

 

While 2,3-BPG at m/z 264.94 marks intra-vascular structure due to its relationship with red blood cells, metabolites such as uridine diphosphate (UDP)-glucose at m/z 565.03 and UDP - N-acetylhexosamine (HexNAc) at m/z 606.05 were strongly detected in cortex of the thymus, bone marrow, and adipose tissues. Considering that 2,3-BPG is colocalized with metabolites in circulation which belongs to a humoral factor and UDP-glucose and UDP-HexNac are representing metabolic status of a solid organ, mostly intracellular. While there seems no significant difference in the intensity of 2,3-BPG between DEX treated and non-treated mice, the distribution of 2,3-BPG shown by tMSI is dependent on its background vascular structure (Figure.3).

 

Remark no 2:

Page 8, line 226

Figure 4. MSI derived from purine and pyrimidine metabolites.

The scale of intensity on the numbers is unreadable.

 

As for this suggestion, we have revised figures by color scale and each numbers were described in figure legends instead. The most importantly, we have edited all figures to utilize the universal color scale among all nucleotides imaging.

 

Remark no 3:

Page 9, line 274-277

Figure 5. Visualization of energy charge (EC) status of multiple thoracic organs with or without DEX administration. … EC in the thymic cortex was extremely reduced after DEX administration. In the medulla of DEX-treated thymus, heterogeneous EC distribution was noticeable.

The intensity scale in the color maps is not the same for the control sample and the Dex sample, hence the comparison is difficult.

EC in the thymic cortex of DEX-sample is heterogeneously distributed. Still there are areas with high intensity. Rewrite this sentence.

 

According to this suggestion, we corrected intensity scale in the color maps to be same for the control sample and the Dex sample, hence the comparison is enabled. EC in the thymic cortex of DEX-sample is heterogeneously distributed. Still there are areas with high intensity.

 

Remark no 4:

Page 4, line 180

Distribution of IMP in both control and DEX treated mice were shown in Supplementary Figure.6.What does it tell us? Lack of description of the figure.

 

Thank you for your valuable comment. We have added the following sentences with one extra reference (36). It is very interesting to know that IMP levels were negatively related to the ATP/ADP ratio or energy status of the skeletal muscles of the thorax.

 

This is one of most prominent metabolic effects of systemic corticosteroid therapy which were noticeable in multiple tissues including aortic walls, esophagus and skeletal muscles of the thorax. Of note, it is also detected in the grey matter of the spinal cord (Supplementary Figure.6).

IMP formation is thought to reflect an imbalance between resynthesis and utilization of ATP. In patients with stable chronic obstructive pulmonary disease (COPD), elevated muscular IMP content has been reported in human studies³⁶. IMP levels were negatively related to the ATP/ADP ratio, suggesting an imbalance between ATP utilization and resynthesis. This is totally consistent with the current study because shift in energy status of thoracic muscle shown in Figure.5 was inversely related with shift in IMP distribution in Supplementary Figure.6 in DEX treated mice. The cause and consequences of these disturbances in muscle energy metabolism in COPD patients³⁶ as well as DEX treatment in the current study need further exploration.

Remark no 5:

Page 4, line 193

… in blue colored compartment in Figure.5

Do You refer to Fig. 6?

 

Yes, it is. We have corrected as above.

 

Remark no 6:

Page 5, line 198

Figure.4

Do You refer to Fig. 4 here (Figure 4. MSI derived from purine and pyrimidine metabolites)?

 

Yes, it is. We have corrected as above.

Author Response File: Author Response.pdf

Reviewer 2 Report

This research described that new imaging method called tMSI. This method revealed that dynamic shift in energy status of multiple thoracic organs after administration of DEX. In addition, whole section imaging gives us wide range information of body change.

The result of metabolic effects of corticosteroid administration in mice were interested.

Thus, totally, I do recommend its publication in Applied Sciences, however it has minor contents.

 

Minor comments

Authors had better described target m/z (ie, ADP, ATP,.,) in manuscript not supplementally data to gives easy understanding for broad reader.

 

Authors described that ‘To our surprise, ATP can be visualized with current protocol without being decayed as post mortem changes.’. Do you have a data by conventional method. Is it true that shortens time of preparation could avoid to decreased ATP signal?

 

Color intensity bar should be added in MSI data.

 

In Fig. 8, m/z 625.16 is interested marker of DEX. At this time, Do authors have idea what kind of species that affected in body?

Author Response

For reviewer2

 

Minor comments

Authors had better described target m/z (ie, ADP, ATP,.,) in manuscript not supplementally data to gives easy understanding for broad reader.

 

We would like to respect above suggestion and have revised our manuscript as abovementioned way in the text.

 

Authors described that ‘To our surprise, ATP can be visualized with current protocol without being decayed as post mortem changes.’. Do you have a data by conventional method. Is it true that shortens time of preparation could avoid to decreased ATP signal?

 

This suggestion is totally valid. So we have rewrote these sentences as follows:

 

As is often the case with MALDI-MSI, metabolites such as ATP will decay rapidly as post mortem (PM) changes within 1 min after death of the animal. A key issue to resolve is prevention of the PM enzymatic degradation of metabolites during the IMS sample preparation process. Our method of tMSI is superior whole-body imaging technology which saves time for sample collection and subsequently freezing in comparison with conventional method.

 

Color intensity bar should be added in MSI data.

 

We appreciate this suggestion and have edited all figures to add universal color intensity bar.

 

In Fig. 8, m/z 625.16 is interested marker of DEX. At this time, Do authors have idea what kind of species that affected in body?

 

We really appreciate this comment. At this moment, we are also thinking that the ion at m/z 625.16 is to be further analyzed for its chemical identity as well as its significance in terms of DEX treatment. This will be realized in the future study. Again, we really appreciate for your concerns.