Effects of brain-derived mitochondria

on the function of neuron and vascular

endothelial cell after traumatic brain
injury. Baoliang Zhang & Yalong Gao &
Qifeng Li & Dongdong Sun & Xinlong
Dong & Xiaotian Li & Wenqiang Xin &
Jianning Zhang
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Effects of brain-derived mitochondria on the function of neuron and vascular

endothelial cell after traumatic brain injury.

Baoliang Zhang, Yalong Gao, Qifeng Li, Dongdong Sun, Xinlong Dong, Xiaotian Li,
Wenqiang Xin, Jianning Zhang

PII: S1878-8750(19)33022-0
DOI: https://doi.org/10.1016/j.wneu.2019.11.172
Reference: WNEU 13841

To appear in: World Neurosurgery

Received Date: 5 September 2019

Revised Date: 28 November 2019
Accepted Date: 29 November 2019

Please cite this article as: Zhang B, Gao Y, Li Q, Sun D, Dong X, Li X, Xin W, Zhang J, Effects of brain-
derived mitochondria on the function of neuron and vascular endothelial cell after traumatic brain injury.,
World Neurosurgery (2020), doi: https://doi.org/10.1016/j.wneu.2019.11.172.

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Effects of brain-derived mitochondria on the function of neuron and vascular
endothelial cell after traumatic brain injury.

Baoliang Zhang12*, Yalong Gao34*, Qifeng Li34*, Dongdong Sun5, Xinlong Dong34,
Xiaotian Li34, Wenqiang Xin34, Jianning Zhang34#

1. Department of General medicine, Tianjin Medical University General Hospital, 154

Anshan Road, Tianjin 300052, China
2. Department of Medical cosmetology, Airport hospital, Tianjin Medical University
General Hospital, 154 Anshan Road, Tianjin 300052, China
3. Department of Neurology, Tianjin Medical University General Hospital, 154
Anshan Road, Tianjin 300052, China
4. Key Laboratory of Post-Neurotrauma Neurorepair and Regeneration in Central
Nervous System, Ministry of Education in China and Tianjin Neurological Institute,
Tianjin, 30052, China
5. Department of Neurology, Tianjin Huanhu Hospital, NO.6 Jizhao Road, Tianjin
300350, China
* The authors contributed equally to the paper.
# The author is corresponding author.

Correspondence: Jianning Zhang

Department of Neurosurgery, Tianjin Medical University General Hospital, 154
Anshan Road, Tianjin 300052, China
Tel +8618526201161
Fax 86 022-60817448
Email: jianningzhang.tmu@gmail.com
 1 Introduction:
2 Traumatic brain injury (TBI) is one of the common causes of high mortality and
3 disability worldwide, especially among the young 1. TBI results in an unfolding
4 sequence of injury to the central nervous system (CNS), and accounts for serious
5 health problems worldwide. There are about 69 million individuals worldwide may
6 suffer fromTBI each year 2. In addition to primary damage, TBI also causes secondary
7 injury including oxidative stress, inflammation, mitochondrial dysfunction, and
8 apoptosis which occurs within short time and may last from hours to days even
9 months 3. These pathological changes affect each other and develop concurrently.
10 Two recent studies demonstrated that metabolic inhibition is associated with the
11 secondary pathology of both pre-clinical and clinical TBI 3, 4. Another study
12 demonstrated that continuous neuroinflammation and disruptions in brain energy
13 metabolism occured frequently in the brain of TBI and the suppression of
14 neuroinflammation and regulation of energy homeostasis are related with an
15 enhancement in cognitive function in the brain 5. A study confirms that cellular
16 energy crisis in the injured brain does exist 6. But, because of multipathological
17 changes after brain injury, the definite molecular mechanisms remain to be fuy
18 explained.
19 With the deeper understanding of mitochondrial function, it has been found that
20 mitochondrial dysfunction plays a subtle role in the disease and gradually attracts
21 more attention 7. Mitochondria are organelles that are widely distributed inside the
22 cytoplasm and function as a source of energy in the metabolic activities of cells. After
23 a TBI, mitochondrial dysfunction can occur regularly and release excessive reactive
24 oxygen species (ROS) and activate the inflammatory pathway 8. ROS are byproducts
25 mainly produced by the normal metabolism of mitochondria, and during times of
26 physiological stress, ROS levels can increase significantly and lead to different level
27 cell damage 9. Thus, regarding TBI-induced severity, it is important to note that
28 abnormal metabolism of energy caused by primary and secondary brain injury and
29 functional deficits are interrelated events. Currently, there are four main methods
30 involving interventions in use related to mitochondrial dysfunction in TBI, including
31 hypothermia, Hyperbaric Oxygen, exercise and antioxidant therapy 10. Although these
32 methods have a certain effect, they still cannot yield satisfactory clinical results.
33 Previous studies have reported that certain cell-derived mitochondria can be
34 transferred to damaged cells and have a mitigating effect on certain diseases through
35 certain mechanisms 11, 12. In recent years, a promising study found out a new
36 mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous
37 neuroprotective and neurorecovery mechanisms after stroke 13. This means that
38 mitochondrial transport between cells may be a target for the treatment of CNS
39 diseases. However, knowledge regarding mitochondrial energy metabolism disorder
40 after repetitive traumatic brain injury and, specifically, whether exogenous
41 mitochondria injection would overcome the energy imbalance, remains blurred.
42 However, there is still no mitochondrial transplantation for the treatment of TBI.
43 The primary problem of this study was to find effective ways to extract and identify
44 active mitochondria, and to demonstrate that exogenous mitochondria can be
45 transferred to cells in vitro experiments. Next, to determine the effects of
46 mitochondria injection in TBI, we investigated whether exogenous mitochondria can
47 modulate cellular respiratory function and reduce BBB leakage and improve
48 neurological electrophysiological activity.
49
50 2. Subjects and Methods:
51
52 2.1. Preparation of brain-derived mitochondria
53
54 We obtained the brain-derived mitochondria (BDMt) by using the previously
55 described method and manufacturer's protocol (tissue mitochondrial isolation kit,
56 Beyotime, Shanghai, China). Briefly, mice were anesthetized with 10% chloral
57 hydrate (3 mL/kg, intraperitoneal injection) and perfused transcardially with cold
58 PBS. Brains were collected and homogenated for 6 times in mitochondrial lyses
59 buffer A on ice by tissue homogenizer. The homogenate was centrifuged at 600g for
60 10 min. Then the supernatant was collected and centrifuged again at 3500g for 10
61 min. In order to obtain high-purity mitochondria, 0.5 ml of 80% percoll, 1.5 ml of
62 52% percoll, 1.5 ml of 26% percoll were sequentially added to the ultracentrifuge
63 tube, and the uppermost layer was crudely extracted mitochondria, and then
64 centrifuged at 20,000 rpm for 45 min. The above centrifugation is carried out at 4 °
65 C.At last, the target product is located between 26% and 52% percoll.
66 To evaluate the activity of isolated mitochondria, the mitochondria was stained
67 with mitochondrial specific fluorescent dye MitoTracker Green (ThermoFisher
68 Scientific, USA), a membrane-potential-independent green fluorescent dye that stains
69 mitochondria in live cell 14. Then analyze the change of fluorescence intensity by flow
70 cytometry. The mitochondrial membrane potential was detected by JC-1 assay kit
71 (Beyotime, Shanghai, China) using microplate reader (Varioskan flash, ThermoFisher
72 Scientific, USA).
73
74 2.2. Experimental design and mitochondria injection
75
76 A total of 80 mice were randomly assigned to the following four groups: sham +
77 saline group (n = 20); TBI + mitochondria group (n = 20); TBI + saline group (n =
78 20); TBI + vehicle group (n = 20). Proper measures were taken to minimize the
79 number of mice used and the pain or discomfort they might experience. Ten minutes
80 after the CCI model, the mice were positioned according to the coordinates: 1.0 mm
81 posterior to bregma, 2.0 mm lateral to sagittal suture, 1.5 mm beneath the surface of
82 brain slowly injected mitochondria at an injection rate of 1 µl/min (10ul) ipsilaterally
83 into injury area and then leaving the needle for 5 min to to prevent backflow.
84 Meanwhile, the sham +saline group and the TBI + saline group and TBI +vehicle
85 group were done with volume-matched saline or storage buffer, instead. Then, four
86 groups of mice were euthanized and the brain were collected in established time.
87
88 2.3. Animal
89
90 Adult male C57BL/6 mice (8–10 weeks old, 20–25 g) were purchased from the
91 Experimental Animal Laboratories of the Academy of Military Medical Sciences
 92 (Beijing, China). All procedures were conducted in strict accordance with the
93 ARRIVE Guidelines (Animal research: reporting of in vivo experiments) and
94 approved by the Ethics Committee of Tianjin Medical University (Tianjin, China).
95 The mice were housed in animal facilities under a standardized 12 h light/dark cycle
96 with controlled temperature and humidity and had free access to food and water. All
97 efforts were made to minimize the number of mice used and their suffering. In all
98 experiments, data were obtained by investigators blinded to the experimental design.
99
100 2.4. Animal model of TBI
101
102 TBI was induced using a controlled cortical impact (CCI) device (eCCI-6.3 device,
103 Custom Design & Fabrication, USA). Briefly, mice were anesthetized with 10%
104 chloral hydrate (3 mL/kg, intraperitoneal injection) and then positioned in a
105 stereotaxic frame (RWD Life Science, Shenzhen, China) with a heating pad to
106 maintain body temperature. After the scalp and fascia were retracted, a 3.5-mm
107 diameter hole was drilled on the right cerebral hemisphere (2.0 mm posterior from
108 bregma and 2.0 mm lateral to the sagittal suture) to expose the intact dura. For
109 moderate-TBI induction, a 3-mm-flat impactor tip was used to impact the exposed
110 dura (impact parameters: velocity: 5.0 m/s, depth: 1.2 mm, dwell time: 200 ms). After
111 CCI injury, the scalp incision was sutured, and the mice were placed in a heating pad
112 until anesthesia recovery. Sham group mice received all of these procedures, but did
113 not receive a CCI.
114
115 2.5. Immunofluorescence microscopy for cells and brain sections
116
117 bEnd3 and PC-12 cells are seeded on microscope coverslips and then treated with
118 OGD following the protocol below. The medium was changed to Dulbecco's modified
119 Eagle's medium (DMEM) without glucose and serum. Hypoxia was induced by
120 placing the cells in a chamber (Billups–Rothenberg) and perfusing the chamber with
121 94% N2, 1% O2 and 5% CO2 for 30 min. After the cells are put in, the chamber was
122 then sealed and kept at 37 °C lasting 6h.We collected Bend3 and PC-12 cells
123 co-cultured with the BDMts respectively. Cells were fixed with 4% formaldehyde for
124 20–30 min, permeabilized with 0.4% Triton X-100 in PBS for 10–20 min, and
125 blocked with 10% donkey serum in PBS for 1 h at room temperature. Cells were then
126 incubated with primary antibody (diluted with 1% donkey serum in PBS) overnight at
127 4 °C and fluorescence-labeled secondary antibody (ZSGB-BIO, 1:500, china) at room
128 temperature for 1 h the next day.
129
130 Brain cryosections were fixed with acetone at −20 °C for 20 min and incubated in
131 3% bovine serum albumin (BSA, SigmaAldrich, USA) for 1 h at 37 °C to block
132 nonspecific staining. The sections were then incubated with the following antibodies
133 at 4 °C overnight: anti-MAP-2 (1:500, abcam, USA), and anti-ZO-1 (1:100,
134 Invitrogen) antibodies. Thereafter, the cryosections were rinsed with PBS and
135 incubated with corresponding secondary antibodies (Alexa Fluor 594: donkey
136 anti-goat; Invitrogen) for 1 h at RT. Finally, nuclei were counterstained (DAPI,
137 Abcam). Images of each section were captured using a fluorescence microscope
138 (Olympus IX81, Tokyo, Japan), and the data were analyzed from 9 randomly selected
139 microscopic fields (three fields per section × three sections per mouse) with NIH
140 ImageJ software. The results were expressed as the proportion of the immunoreactive
141 area. All analyses were performed in a blinded manner.
142
143 2.6. Western blotting
144
145 Western blot analysis was performed as previously described [11]. The PVDF
146 membranes were respectively incubated with rabbit anti-ZO-1 (1:200, Abcam), rabbit
147 anti-Occludin (1:300, Abcam), rabbit anti-GAP43 (1:500, CST), rabbit
148 anti-Synapsin-I (1:500, CST) and rabbit anti-β-actin (1:500, CST) at 4°C overnight.
149 Then, the membranes were incubated with the appropriate HRP-conjugated secondary
150 antibodies (ZSGB-BIO, china) for 2 h. Enhanced
151 chemiluminescence luminol reagent (Beyotime, Shanghai, China) was used for
152 protein quantity determination. The densitometric analysis of the target protein bands
153 were analyzed using ChemiDoc™ XRS+ Imaging System (Bio-Rad). The data reflect
154 the intensity of the target protein band, which was normalized to the intensity of the
155 endogenous control of each sample (expressed in arbitrary units). Measurement of
156 mean pixel density of each band was detected using the Quantity One.software
157 (Bio-Rad).
158
159 2.7. Brain water content
160
161 Brain water content was measured by the wet-dry weight method at 72 h following
162 TBI, as previously reported 15. Briefly, brains were obtained without transcardiac
163 perfusion, after which their weights were determined by an electronic analytical
164 balance. The brains were subsequently dried in an electrothermostatic blast oven for
165 72 h at 60◦C, after which their dry weights were determined by the same electronic
166 analytical balance. Brain water content (%) was calculated as (wet brain weight − dry
167 brain weight)/wet brain weight × 100%.
168
169 2.8. Evans blue permeability assay
170
171 Evans blue (EB) dye extravasation was used to evaluate BBB permeability as
172 previously described 16. Briefly, mice were injected with 100 µl of 4% Evans blue
173 solution (EB) dye 3 d after TBI. One hour later, animals were perfused with PBS for 4
174 min, and the brains were removed and separated into the ipsilateral core region of
175 TBI. Each hemisphere was then dissected, weighed, and homogenized in
176 N,N-dimethylformamide (Sigma-Aldrich, USA) and centrifuged for 50 min at 14,000
177 r.p.m. (Thermo Biofuge, D37520 Osterode, Germany). The supernatants were
178 collected, and quantification of EB extravasation was determined from the formula:
179 (A620nm − ((A500nm + A740nm)/2)) per g wet weight. Background EB levels in the
180 contralateral region was subtracted from that of the ipsilateral core region.
181
182 Statistical analysis
183 Each experiment was repeated three times and the results are expressed as the
184 means ± SD. One-way analysis of variance or Student’s t-test and the least signifcant
185 difference were used to infer signifcant differences in the data. SPSS 13.5 covariance
186 software for Windows (SPSS, Inc., Chicago, IL, USA) was used to perform these
187 analysis. P<0.05 was considered to indicate a statistically signifcant difference. All
188 values are expressed as the mean ± standard deviation.
189
190 Results:
191
192 3.1. Mitochondrial identification, labeling and number
193
194 According to the kit's instructions (Beyotime, Shanghai, China), we extracted
195 mitochondria from fresh brain tissue. Electron microscopy confirmed that the
196 mitochondrial structure derived from fresh brain tissue is intact compared with frozen
197 brain tissue and mitochondrial cristae is clearly visible (Figure1A, B).
198 Mitotracker-labeling suggested that these mitochondria may still be functional in
199 maintaining membrane potential. Flow cytometry (the lower right quadrant represents
200 mitotracker positive mitochondria) showed that the concentration of mitochondria is
201 approximately 1.1*10^7/ul (Figure1C). Briefly, this method of extracting
202 brain-derived mitochondria is reliable.
203
204 3.2. Mitochondrial function detection of brain tissue
205
206 As previously mentioned, mitochondrial activity is the key to treatment. We
207 evaluated mitochondrial membrane potential including frozen and thawed tissue and
208 fresh tissue. By comparison, freeze-thaw induced loss of mitochondrial membrane
209 potential (Figure2A). However, the mitochondrial membrane potential of fresh tissue
210 showed larger internal and external voltage difference (Figure2B). There is a
211 statistically significant difference between the membrane potential of mitochondria
212 from two different tissue sources (Figure2C). Besides, we also performed
213 mitochondrial RCR [respiratory control ratio (state 3/state 4)] to determine the
214 differences in the tissue source (Figure2D, E). Fresh and frozen-thawed tissue showed
215 significant statistical differences in respiratory state 3 (Figure2F). Finally, we
216 concluded that the mitochondrial physiological function of fresh tissue sources is
217 complete (Figure2G).
218
219 3.3 Mitochondria improve nerve cell function in vitro
220
221 We obtained the results that exogenous mitochondria could be uptake by PC12
222 cells by fluorescence microscopy. Briefly, we pre-stained the mitochondria with the
223 specific fluorescent dye mitotracker (green), then co-cultured with PC12 cells after
224 which experienced OGD treatment. We observed some green spots inside PC12 cells,
225 which stained by MAP-2(red) to mark the cytoskeleton. The results indicated that
226 some mitochondria could enter into damaged cells (Figure3 A). And the
227 mitochondrial uptake rate by the PC12 cells is 15% approximately. The discovery of
228 this phagocytic phenomenon explains the mechanism of mitochondrial therapeutic
229 effects to some extent. Moreover, after mitochondria were uptake by PC12 cells, we
230 observed that the respiratory function of the damaged cells changed by RCR. The
231 results showed that the improvement of OGD cells changed at 52 min after co-culture
232 with brain derived mitochondria and there was a statistical difference between the
233 mitochondrial treatment group and the OGD group (Figure3 E). Briefly, for the
234 mitochondrial intervention group, Oxygen Consumpt Rate (OCR) increased by 68%
235 in PC-12 cells, while the other control groups did not change significantly. Synaptic
236 plasticity related proteins, for example synapsin-I and GAP-43 are closely related to
237 synaptic plasticity of neuronal cells. Hypoxia and hypoglycemia can led to remarkable
238 synaptic plasticity-related proteins loss in PC-12 cells, however, the application of
239 exogenous mitochondria can reverse this trend (Figure3 B). Statistical results show
240 that GAP43 (Figure3 C) and synapsin-I (Figure3 D) expression levels are
241 significantly different between the mitochondria treatment group and the OGD group
242 (P<0.05).
243
244 3.4. Mitochondria administration promoted angiogenesis in vivo
245
246 The tissue repair is associated with mitochondrial energy metabolism disorder after
247 trauma. We therefore investigated the effect of mitochondria local injection on
248 vascular endothelial regeneration. On the 7th day after TBI, neovascular density in the
249 injured area is determined by CD31 through immunofluorescence, which is vascular
250 endothelial cells marker (Fig. 5A). We found that in the mitochondrial intervention
251 group, neovascularization was significantly increased while in the TBI group
252 neovascularization is less on the 7th day (Fig. 5B p<0.05).
253
254 3.5. Mitochondria administration reduced BBB damage and brain water content in
255 TBI mice
256
257 BBB damage and brain edema, which peaked at 3 d after TBI, is an important
258 surrogate marker associated with patient prognosis. To determine whether brain
259 tissue-derived mitochondria administration reduces brain edema and BBB leakage, we
260 measured brain water content and EB leakage in each group. The injury group had
261 significantly higher brain water content than the sham group (82.00 ± 0.40 vs 78.50
262 ± 0.30, n=10, p < 0.05). And mitochondria administration significantly reduced
263 brain water content in the mitochondria -treated group compared with the TBI group
264 (79.5 ± 0.60 vs 82.0 ± 0.40, n=10, p < 0.05) (Fig. 5D). The same positive effect of
265 mitochondria was observed between mitochondria injection group and CCI group in
266 the EB leakage experiment (Fig. 5C) (4.20 ± 0.15 vs 7.0 ± 0.25, n=10, p < 0.05).
267
268 3.6. Mitochondria improve endothelial cells function in vitro and in vivo
269
270 We found that mitochondria could be uptake by bEnd3 cells by fluorescence
271 microscopy. Briefly, pre-stained mitochondria with the mitotracker, co-cultured with
272 bEnd3 cells after which experienced OGD treatment. We observed some green spots
273 inside bEnd3 cells, which stained by CD31 to depict cell contours. The results
274 indicated some mitochondria could enter into damaged cells (Figure4 A) and the
275 uptake rate by the bEnd3 cells is about 12%. After which, we observed that the
276 respiratory function of the damaged cells changed by OCR. In a word, for the
277 mitochondrial intervention group, OCR increased apparently in bEnd3 cells, while the
278 other control groups did not change obviously (Figure4 E). Tight junction proteins
279 (TJPs) are the components that connect endothelium, which sealed the intracellular
280 gaps between bEnd3 and maintain the integrity of endothelial barrier 17. Currently, the
281 most important proteins contributing to tight junction formation are believed to be
282 ZO-1 and Claudin-5 18. We quantified their expression levels using WB, and found
283 that their levels were decreased after OGD treatment (Figure4 B). However, in the
284 brain-derived mitochondria treatment group, the expression of ZO-1 (Figure4 C) and
285 Occludin (Figure4 D) was significantly increased and there was a statistically
286 significant difference between the experimental group and the OGD group (P<0.05).
287 This means that TJPs could be up-regulated by exogenous mitochondria co-culture.
288
289 3.7. Mitochondria administration improved LTP
290
291 As previously demonstrated, long-term depression (LTD, the electrophysiological
292 basis of memory) are associated with synaptic plasticity during learning and memory
19
293 . On the 7th day after CCI injury in mice, the LTD of ipsilateral hippocampus was
294 significantly weakened. However, after the intervention of mitochondria injection, the
295 LTD on the injured side has a significant improvement (Fig. 5E, p<0.05) from 20
296 minutes to 60 minutes.
297
298 Discussion:
299
300 A loss of mitochondrial function influences a number of vital cellular events after
301 TBI 20. Therefore, mitochondrial dysfunction is a very appealing target in TBI models
302 owing to their pivotal significance in both cellular homeostasis and their involvement
303 in cell death 21. Although literature reports that some drugs have a good effect on
304 animal models, the FDA has not approved any effective drug for the treatment of TBI
305 until now. Thus, in this study, we investigated the neuroprotective effects of
306 exogenous mitochondria in mice subjected to TBI. We found that mitochondria
307 application in vitro and in vivo reduce apoptosis and improved neurologic outcomes
308 after TBI. Furthermore, we uncovered convincing evidence indicating that exogenous
309 mitochondria can attenuate BBB leakage by inhibiting tight junction protein (TJP)
310 destruction.
311 In vitro, mitochondria can be engulfed by OGD-treated Bend3 and PC-12 cells with
312 an average intake rate of 12% and 15%, respectively. However, it is still unknown
313 how the free mitochondria entered the cell. A previous study showed that bone
314 marrow-derived stromal cells (mBMSCs) formed connexin 43 (Cx43)-containing gap
315 junctional channels (GJCs) with the alveolar epithelia in mice, releasing
316 mitochondria-containing microvesicles that the epithelia engulfed. Recent research
317 reported that astrocytes released extracellular mitochondria particles through a
318 calcium-dependent mechanism involving CD38/cyclic ADP ribose signaling 13.
319 Different from this experiment is that mitochondria are transferred in the form of
320 mitochondrial particles in the above study. Thus, the mechanism by which free
321 mitochondria migrate into cells remains to be further explored. Moreover, there is an
322 urgent need to further discover the phagocytosis when injecting mitochondria in vivo.
323 Many TBI survivors sustain neurological disability and cognitive impairments due
324 to the lack of defined therapies to decrease TBI-derived secondary BBB breakdown 3.
325 Tight junction protein (TJP) are the hallmark of BBB integrity that necessarily
326 contributes to its structural intact. The paracellular flux of hydrophilic molecules
327 across the BBB is thereby limited which is essential for the functional integrity of the
328 CNS 22. Our study found the fact that the expression of ZO-1 and occludin is
329 significantly increased in damaged Bend3 cells after co-incubation with exogenous
330 mitochondria, compared with OGD cells. Moreover, the effect of this exogenous
331 mitochondria on the insulted BBB induced by TBI has also been similarly repeated in
332 vivo. These results may suggest that the energy metabolism disorder of endothelial
333 cells affects the synthesis and maintenance of TJPs. Subsequently, the specific energy
334 metabolic mechanisms involved need to be further explored.
335 GAP-43 which is considered to be the intracellular growth associated protein, plays
336 an essential role in neuron growth during brain development and neuroregeneration
337 after TBI 23-25. Frequently, GAP-43 can be used as a marker of nerve regeneration and
338 recovery. The recognition that neurogenesis is closely related to mitochondrial energy
339 metabolism has spun off research towards the recovery of neurological function
340 through mitochondrial transplantation 26. In our experiment, oxygen-glucose
341 deprivation down-regulated the expression of GAP-43 protein in PC-12 cells, but
342 exogenous mitochondria could reverse the content of this protein. There are reports
343 that neurogenesis required much energy consumption, brain depends on aerobic
344 energy metabolism, hence on mitochondrial activity 27, 28. Consistent with these
345 studies, the present study found that increased cellular energy metabolism contributes
346 to the expression of nerve regeneration related proteins. Some important nerve growth
347 factors, such as synapsin I, had a similar change in our OGD model experiment in
348 PC-12 cells.
349 The fact that TBI animals impairs mitochondrial respiration, leads to reactive
350 oxygen species (ROS) overproduction, and neuronal apoptosis is responsible for the
351 neurological deficits29. Although a variety of studies have found that TBI can causee
352 neuronal cell apoptosis and neurological deficits, the detailed mechanisms explaining
353 TBI associated neurodegeneration have not been fully clarified. A recent study
354 showed that fragmented mitochondria were increased 24–48 h after injury in primary
355 hippocampal neurons 28. But, our study lack of the result that whether mitochondria
356 transplantation could reduce overall apoptosis 3 days after TBI. However, previous
357 research concluded that transfer of functional mitochondria reverse stressed cells in
358 the early stages of apoptosis 30. Moreover, the phenomenon that mitochondrial
359 transplantation can reduce apoptosis has been observed in other diseases 31.In short, it
360 can be inferred from the above conclusions that mitochondrial transplantation may
361 have positive role in reducing apoptosis.
362 The BBB is a unique and integral feature of the central nervous system,
363 encompassing astrocytic processes, pericytes and the adjacent neurons in addition to
364 capillaries 32. After TBI, BBB experienced a biphasic disruption. In addition to the
365 primary insult for microvessels caused by mechanical force, secondary injury, such as
366 tight junction complexes destruction, play a major role in the subsequent evolution of
367 damage 33, 34. After the stroke occurs, the destruction and pathological changes of
368 BBB are closely related to the energy metabolism disorder caused by mitochondria 35.
369 These studies provide a basis for research on mitochondrial transplantation for TBI.
370 After all, the relationship between BBB destruction and mitochondrial energy
371 metabolism is not clear enough in TBI. In our research, mitochondrial transplantation
372 can relieve brain edema and reduce BBB leakage, which shows that the “energy
373 plant” of the cockroach plays a subtle role in the destruction of BBB. Exogenous
374 mitochondria transplantation could alleviate excessive inhibition of LTP in the
375 hippocampus on the ipsilateral side of the injury 7 days after TBI. This shows its
376 beneficial effect on the recovery of nerve function. However, one thing that puzzles
377 us is that mitochondria injection alleviated undue suppression of LTP as a result of
378 improving BBB or its direct neuroprotection. Because the function and activity of
379 neurons are affected by the integrity of the BBB 36.
380 In the past few decades, various models of mitochondrial transplantation have been
381 investigated, each with their own caveats and insights, and these practices provided a
382 good reference for our application on the human37. As early as 1997 and 1998, Cohen
383 et al. reported that cytoplasmic transfer therapy was tested in the clinic to transfer the
384 cytoplasm of a healthy donor oocyte into a compromised oocyte, which was then
385 fertilized and implanted and resulted in successful pregnancy38, 39. Mitochondria are
386 contained in cytoplasmic. Reardon found that mitochondrial transplantation can be
387 used to treat embryos with mitochondrial gene defects40. Up to date, mitochondrial
388 replacement therapy includes both in vivo and in vitro methods. There are some
389 studies on mitochondrial transplantation for human in vitro treatment during few
390 decades.But the use of mitochondrial transplantation for human in vivo is currently
391 rare. However, the fact that therapeutic effects of mitochondrial transplantation have
392 many good applications in animal models may let us believe that applications in the
393 human body may receive compelling results. In fact, the worry what we have to face
394 is that while mitochondria transfer therapy resulted in successful births, it was banned
395 in some countries for ethical reasons.
396 However, our study contained some limitations. The secondary damage associated
397 with TBI primarily include energy metabolism dysfunction, ER stress, Ca2+ overload,
398 oxidative stress, excitotoxicity and free-radical excess, all of which can lead to BBB
399 disruption and apoptosis 41, 42. From the perspective of energy metabolism of
400 mitochondria, we studied the effects of mitochondrial transplantation on the
401 improvement of cellular respiration in vitro and attenuation effect of BBB leakage in
402 vivo, but did not investigate its downstream signaling proteins after TBI. Further
403 studies are required to determine the mitochondrial transfer mechanism in vivo and its
404 effects on other pathological changes.
405
406 References:
407 1. Dong T, Zhi L, Bhayana B, Wu MX. Cortisol-induced immune suppression by
408 a blockade of lymphocyte egress in traumatic brain injury. J
409 Neuroinflammation. 2016;13:197
410 2. Dewan MC, Rattani A, Gupta S, Baticulon RE, Hung YC, Punchak M, et al.
411 Estimating the global incidence of traumatic brain injury. Journal of
412 neurosurgery. 2018:1-18
413 3. Wang ZG, Cheng Y, Yu XC, Ye LB, Xia QH, Johnson NR, et al. Bfgf protects
414 against blood-brain barrier damage through junction protein regulation via
415 pi3k-akt-rac1 pathway following traumatic brain injury. Mol Neurobiol.
416 2016;53:7298-7311
417 4. Cassidy JD, Carroll LJ, Peloso PM, Borg J, von Holst H, Holm L, et al.
418 Incidence, risk factors and prevention of mild traumatic brain injury: Results
419 of the who collaborating centre task force on mild traumatic brain injury.
420 Journal of rehabilitation medicine. 2004:28-60
421 5. Briones TL, Woods J, Rogozinska M. Decreased neuroinflammation and
422 increased brain energy homeostasis following environmental enrichment after
423 mild traumatic brain injury is associated with improvement in cognitive
424 function. Acta neuropathologica communications. 2013;1:57
425 6. Hill JL, Kobori N, Zhao J, Rozas NS, Hylin MJ, Moore AN, et al. Traumatic
426 brain injury decreases amp-activated protein kinase activity and
427 pharmacological enhancement of its activity improves cognitive outcome.
428 Journal of neurochemistry. 2016;139:106-119
429 7. Chan DC. Mitochondria: Dynamic organelles in disease, aging, and
430 development. Cell. 2006;125:1241-1252
431 8. Abdul-Muneer PM, Chandra N, Haorah J. Interactions of oxidative stress and
432 neurovascular inflammation in the pathogenesis of traumatic brain injury. Mol
433 Neurobiol. 2015;51:966-979
434 9. Kostyuk VA, Potapovich AI. Mechanisms of the suppression of free radical
435 overproduction by antioxidants. Frontiers in bioscience (Elite edition).
436 2009;1:179-188
437 10. Hiebert JB, Shen Q, Thimmesch AR, Pierce JD. Traumatic brain injury and
438 mitochondrial dysfunction. The American journal of the medical sciences.
439 2015;350:132-138
440 11. Islam MN, Das SR, Emin MT, Wei M, Sun L, Westphalen K, et al.
441 Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary
442 alveoli protects against acute lung injury. Nature medicine. 2012;18:759-765
443 12. Li X, Zhang Y, Yeung SC, Liang Y, Liang X, Ding Y, et al. Mitochondrial
444 transfer of induced pluripotent stem cell-derived mesenchymal stem cells to
445 airway epithelial cells attenuates cigarette smoke-induced damage. American
446 journal of respiratory cell and molecular biology. 2014;51:455-465
447 13. Hayakawa K, Esposito E, Wang X, Terasaki Y, Liu Y, Xing C, et al. Transfer
448 of mitochondria from astrocytes to neurons after stroke. Nature.
449 2016;535:551-555
450 14. Poot M, Zhang YZ, Kramer JA, Wells KS, Jones LJ, Hanzel DK, et al.
451 Analysis of mitochondrial morphology and function with novel fixable
452 fluorescent stains. The journal of histochemistry and cytochemistry : official
453 journal of the Histochemistry Society. 1996;44:1363-1372
454 15. Yan EB, Hellewell SC, Bellander BM, Agyapomaa DA, Morganti-Kossmann
455 MC. Post-traumatic hypoxia exacerbates neurological deficit,
456 neuroinflammation and cerebral metabolism in rats with diffuse traumatic
457 brain injury. J Neuroinflammation. 2011;8:147
458 16. Gao W, Zhao Z, Yu G, Zhou Z, Zhou Y, Hu T, et al. Vegi attenuates the
459 inflammatory injury and disruption of blood-brain barrier partly by
460 suppressing the tlr4/nf-kappab signaling pathway in experimental traumatic
461 brain injury. Brain Res. 2015;1622:230-239
462 17. Haseloff RF, Dithmer S, Winkler L, Wolburg H, Blasig IE. Transmembrane
463 proteins of the tight junctions at the blood-brain barrier: Structural and
464 functional aspects. Seminars in cell & developmental biology. 2015;38:16-25
465 18. Thal SC, Neuhaus W. The blood-brain barrier as a target in traumatic brain
466 injury treatment. Archives of medical research. 2014;45:698-710
467 19. He Y, Kulasiri D, Samarasinghe S. Modelling bidirectional modulations in
468 synaptic plasticity: A biochemical pathway model to understand the
469 emergence of long term potentiation (ltp) and long term depression (ltd).
470 Journal of theoretical biology. 2016;403:159-177
471 20. Wang WX, Sullivan PG, Springer JE. Mitochondria and microrna crosstalk in
472 traumatic brain injury. Progress in neuro-psychopharmacology & biological
473 psychiatry. 2017;73:104-108
474 21. Yonutas HM, Vekaria HJ, Sullivan PG. Mitochondrial specific therapeutic
475 targets following brain injury. Brain Res. 2016;1640:77-93
476 22. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and
477 function of the blood-brain barrier. Neurobiology of disease. 2010;37:13-25
478 23. Clayton GH, Mahalik TJ, Finger TE. Expression of gap43 mrna in normally
479 developing and transplanted neurons from the rat ventral mesencephalon. The
480 Journal of comparative neurology. 1994;347:470-480
481 24. Benowitz LI, Routtenberg A. Gap-43: An intrinsic determinant of neuronal
482 development and plasticity. Trends in neurosciences. 1997;20:84-91
483 25. Denny JB. Molecular mechanisms, biological actions, and neuropharmacology
484 of the growth-associated protein gap-43. Current neuropharmacology.
485 2006;4:293-304
486 26. Wang Y, Ni J, Gao C, Xie L, Zhai L, Cui G, et al. Mitochondrial
487 transplantation attenuates lipopolysaccharide- induced depression-like
488 behaviors. Progress in neuro-psychopharmacology & biological psychiatry.
489 2019;93:240-249
490 27. Colangelo AM, Cirillo G, Alberghina L, Papa M, Westerhoff HV. Neural
491 plasticity and adult neurogenesis: The deep biology perspective. Neural
492 regeneration research. 2019;14:201-205
493 28. Chien L, Liang MZ, Chang CY, Wang C, Chen L. Mitochondrial therapy
494 promotes regeneration of injured hippocampal neurons. Biochimica et
495 biophysica acta. Molecular basis of disease. 2018;1864:3001-3012
496 29. Fischer TD, Hylin MJ, Zhao J, Moore AN, Waxham MN, Dash PK. Altered
497 mitochondrial dynamics and tbi pathophysiology. Frontiers in systems
498 neuroscience. 2016;10:29
499 30. Wang X, Gerdes HH. Transfer of mitochondria via tunneling nanotubes
500 rescues apoptotic pc12 cells. Cell death and differentiation.
501 2015;22:1181-1191
502 31. Perez GI, Acton BM, Jurisicova A, Perkins GA, White A, Brown J, et al.
503 Genetic variance modifies apoptosis susceptibility in mature oocytes via
504 alterations in DNA repair capacity and mitochondrial ultrastructure. Cell death
505 and differentiation. 2007;14:524-533
506 32. Prakash R, Carmichael ST. Blood-brain barrier breakdown and
507 neovascularization processes after stroke and traumatic brain injury. Current
508 opinion in neurology. 2015;28:556-564
509 33. Rodriguez-Baeza A, Reina-de la Torre F, Poca A, Marti M, Garnacho A.
510 Morphological features in human cortical brain microvessels after head injury:
511 A three-dimensional and immunocytochemical study. The anatomical record.
512 Part A, Discoveries in molecular, cellular, and evolutionary biology.
513 2003;273:583-593
514 34. Sangiorgi S, De Benedictis A, Protasoni M, Manelli A, Reguzzoni M, Cividini
515 A, et al. Early-stage microvascular alterations of a new model of controlled
516 cortical traumatic brain injury: 3d morphological analysis using scanning
517 electron microscopy and corrosion casting. Journal of neurosurgery.
518 2013;118:763-774
519 35. Nguyen H, Zarriello S, Rajani M, Tuazon J, Napoli E, Borlongan CV.
520 Understanding the role of dysfunctional and healthy mitochondria in stroke
521 pathology and its treatment. International journal of molecular sciences.
522 2018;19
523 36. Chauhan NB. Chronic neurodegenerative consequences of traumatic brain
524 injury. Restorative neurology and neuroscience. 2014;32:337-365
525 37. Gollihue JL, Rabchevsky AG. Prospects for therapeutic mitochondrial
526 transplantation. Mitochondrion. 2017;35:70-79
527 38. Cohen J, Scott R, Schimmel T, Levron J, Willadsen S. Birth of infant after
528 transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet
529 (London, England). 1997;350:186-187
530 39. Cohen J, Scott R, Alikani M, Schimmel T, Munne S, Levron J, et al.
531 Ooplasmic transfer in mature human oocytes. Molecular human reproduction.
532 1998;4:269-280
533 40. Rebbeck CA, Leroi AM, Burt A. Mitochondrial capture by a transmissible
534 cancer. Science (New York, N.Y.). 2011;331:303
535 41. Faridar A, Bershad EM, Emiru T, Iaizzo PA, Suarez JI, Divani AA.
536 Therapeutic hypothermia in stroke and traumatic brain injury. Frontiers in
537 neurology. 2011;2:80
538 42. Pearn ML, Niesman IR, Egawa J, Sawada A, Almenar-Queralt A, Shah SB, et
539 al. Pathophysiology associated with traumatic brain injury: Current treatments
540 and potential novel therapeutics. Cellular and molecular neurobiology.
541 2017;37:571-585
542
 Highlights

Exogenous mitochondria can be uptaken by cell and improve cellular respiration

Mitochondria implantation prevent damage to TJPs and I, improve impaired BBB
after TBI
Mitochondria therapy reduce excessive inhibition of LTP and related to long-term
prognosis in TBI mice
 Author contributions section

Conception and design of the manuscript: JNZ. Drafting of the manuscript: BLZ, YLG, QFL.
Critical revision of the manuscript for important intellectual content DDS, XLD, XTL, WQX :.
Manuscript supervision: BLZ, YLG, QFL. Final approval of the revised manuscript: BLZ, YLG,
QFL.
Abbreviation
TBI = traumatic brain injury; BBB = blood brain barrier; OGD = oxygen glucose
deprivation; CCI = controlled cortical impact; RCR = respiratory control rate; LTP =
long-term depression; CNS = central nervous system; ROS = reactive oxygen species;
BDMt = brain-derived mitochondria; TJPs = Tight junction proteins; PBS= Phosphate
buffered saline; OCR= oxygen consumpt rate; EB= Evans blue;
The authors declare that they have no competing interests.
Another random document with
no related content on Scribd:
 "No, I can't be let off like that. I wouldn't have done that, though it
helped me to decide, of course. But I took him, because I thought I did love
him, and now, after keeping company just on a year, I know I do not. Now
you're a man that understands things."

"Don't you fancy that. None on God's earth is more puzzled about things
than me. I've had a puzzling life I may tell you."

"I haven't. Till now my life's been as clear as sunshine. But now—now
I'm up against a pretty awful thing, and it's cruel hard to make up my mind.
Was you ever really in love?"

"Never mind me."

"Was you ever in doubt, I mean?"

"Never."

"I don't ask for rudeness, but reason. There's nobody you can ask in my
life, because they be all biased. I'm not thinking of myself—God judge me
if I am. I'm just wondering this: Can I be the right down proper good wife
Johnny deserves to have if I don't love him? And the question that's so hard
is, ought I to marry him not loving him? Not because of my feelings, but
because of his future. Think if you was him, and loved a woman as truly as
he loves me, and you had to say whether you'd marry her and chance the
fact she didn't love you, or, knowing she didn't, would give her up."

"That's not how it is, though. Johnny don't know you don't love him. He
don't know what you're feeling. I judge that by what he says, because he
often drops in and talks openly, finding all on his side."

"What would you do?"

"If I wanted to marry a woman and she'd said 'yes,' but afterwards found
herself mistook, I shouldn't love her no more."

"Then you don't know much about love."

"Very likely I don't."

 "It's a selfish thing. If I was in love, I'd be like Johnny—and worse. A
proper tigress I expect."

"Are you in love?"

"No, I swear I'm not. Not with anybody. I've growed up, you see, since I
said 'yes' to John. I was a child, for all my years, when I said it. Growing up
ain't a matter of time; it's a matter of chance. Some people never do grow
up. But I have, and though I don't know what it would be like to fall in love,
I know parlous well I'm not, and never was. And it comes back just to what
I said. Would it be better for Johnny to marry him not loving him, because
I've promised to do so, or would it be better for him if I told him I wasn't
going to? That's the question I've got to decide."

"You'll decide right," he said. "And you don't want other people's views.
You know."

"I know what I'd like to do; but just because my own feeling is strong
for telling him I won't marry him, I dread it. Of course he'll say I'm only
thinking of myself."

"You can't be sure what he'll say."

"Yes, I can: I know him."

"If he knew you didn't love him——"

"He'd only say he'd larn me how to later. But he wouldn't believe it."

"If you was to hold off much longer, he'd chuck you perhaps."

"Never. I'm his life. He says it and he means it."

"But to marry him would be your death?"

She nodded.

"Yes, I think."
 "Perhaps you're wrong, however."

"Very likely. My first thought was to tell him how it was with me and
leave it to him. But I know what he'd do. He'd only laugh at me and not take
it serious, or let me off."

"You are thinking for yourself then?"

"I suppose I am."

"It's natural. You've got your life to live."

"Be sporting," she said. "Don't think of me and don't think of him. Put
us out of your mind and just say what you'd do if you was me."

He felt a little moved for her. It is pathetic to see a resolute creature

reduced to irresolution. The manhood in him inclined Lawrence to take her
part against the man. It seemed an awful thing that her life should be ruined,
as it must be if she married one she did not love. He liked Dinah better than
Johnny, for the latter's arrogance and rather smug and superior attitude to
life at large did not attract Maynard.

"It's never right under any circumstances for a woman to marry a man
she does not love," he said.

"You think so?"

"I do—I'm positive."

"Even if she's promised?"

"Your eyes are opened. You promised because you thought you loved
him. Now you know right well you don't. A proper man ought to bend to
that, however much it hurts. And if you still think it's your duty to marry
him, I say duty's not enough to marry on."

"It's hurting me fearfully, and there's something awful wrong about it.
They want me away from here—Mrs. Bamsey and Jane—that's natural too.
Though why I'm confiding in you I don't know. Something have drove me
to do it. But I know you'll be faithful."

"I wish I could help you, miss. I can only say what I think."

"You have helped me I reckon. You've helped me a lot. I was half in a

mind to go and see Enoch Withycombe; but he's old, and the young turn to
the young, don't they?"

"I suppose they do; though I dare say the old know best, along of
experience."

"The old forget a lot. They always begin by telling you they remember
what it was to be young themselves; but they don't. They can't. Their blood
runs slower; they're colder. They've changed through and through since they
were young. They can't remember some things."

"I dare say they can't."

"Will you come for a walk with me one day and show me that stone you
was telling about—the face?"

"You remember that?"

"Yes; you was going to say more about it the last Sunday you was here;
then you shut up rather sudden."

The idea of a walk with Dinah had certainly never entered Maynard's
head. He remained silent.

"D'you think it would be wrong, or d'you only think it would be a

nuisance?" she asked.

"It's a new notion to me. I'd like to pleasure you and it wouldn't be a
nuisance—far from it I'm sure; but as to whether it would be wrong—it
would and it wouldn't I fancy. It couldn't be wrong in itself; but seeing
you're tokened to another man, you're not free to take walks with Dick,
Tom, or Harry. No doubt you see that."
 "John wouldn't like it?"

"Certainly he wouldn't. You know that."

"Would you mind my walking with another man if you was engaged to
me?"

"Yes, I should, very much indeed; especially if I was in the same fix that
John Bamsey is."

"Poor John. There's such a thing as liking a man too well to love him,
Mr. Maynard."

"Is there?"

"I'm beginning to feel—there—I've wasted enough of your time. You

won't go for a walk with me?"

"I'd like to go for a walk with you."

"I'll ask you again," she said. "Then, whether I marry John, or don't
marry John, there'll be no reason against."

"I quite understand."

"To see that face on the stone. You'll find Mr. Chaffe in his workshop.
Holidays are naught to him. Good-bye. Truth oughtn't to hurt honest people,
ought it?"

"Nothing hurts like truth can, whether you're honest, or whether you're
not."

He went forward turning over with mild interest the matter of the
conversation. He was little moved that she should have asked him to go for
a walk. From any other young woman such a suggestion had been
impressive; but not from her. He had noticed that she was never illusive and
quite unpractised in the art of lure, or wile. The stone he had mentioned was
a natural face carved by centuries of time, on the granite rocks of Hey Tor,
some miles away. He had mentioned it in answer to a remark from
Benjamin Bamsey, and then, for private but sufficient reasons he had
dropped the subject. His connection with the stone belonged to a time far
past, concerning which he was not disposed to be communicative. That she
should have remembered it surprised him. But perhaps the only thing that
had really interested her was the fact he dropped the subject so suddenly.

He fell to thinking on his own past for a time, then returned to Dinah.
That she could confide in him inclined him to friendship. He admired her
character and was sorry for the plight in which she found herself. He hoped
that she might drop Bamsey and find a man she could love. He was aware
that her position in her step-father's house held difficulties, for the situation
had often been discussed at Falcon Farm. Whether she decided for John, or
against him, it was probably certain she would leave Green Hayes; and that
would mean distress for Benjamin Bamsey. He was sorry for all concerned,
but not inclined to dwell over-much on the subject. His own thoughts were
always enough for him, and his experience had tended somewhat to freeze
the sources of charity and human enthusiasm at the fount. He was not
soured, but he was introspective to the extent that the affairs of his fellow
creatures did not particularly challenge him. Thus it was left for Thomas
Palk to see the truth of the situation at Falcon Farm; Lawrence had never
troubled to realise it for himself. It seemed improbable that he would be
woven into the texture of other lives again. Indeed, he had long since
determined with himself that he would never be.

Arthur Chaffe was making a coffin.

"The dead can wait for no man," he said. "A poor old widow; but I'm
under her command for the moment; and she shall have good work."

Lawrence told the matter of the hurdles and Mr. Chaffe promised to do
what he could.

"Joe treats time with contempt," he declared. "He did ought to have told
me long ago; but I always reckon with the likes of him. I think for a lot of
people and save them from their own slow wits. Not that Stockman's got
slow wits. His wits serve him very well indeed, as no doubt you've found."

"He's a good farmer and a kind-hearted sort of man."

 "So he is, so he is. You'll not hear me say a word against him."

"Yet a few do."

"They do. But mind you, when he says he worked as a young man, it's
true. He did work and took a long view, so now you find him as he is. But
he never loved work for itself, same as I do. Work never was meat and
drink to him; and when it had got him what he wanted, he was very well
content to play and let others work for him. And knowing well what work
means, nobody he employs will ever deceive him on the subject."

"He sees that we earn our money. But he's fair."

"Ah! To be fair with your neighbour is a great gift. Few are, and who
shall wonder? Now Joe's a man who takes a generous view of himself. But
'tis better to be hard on yourself and easy with other people—don't you
think?"

"A fine thing, to be hard on yourself, no doubt," admitted Lawrence.

"Yes, and them who are hardest on themselves will often be easiest with
their neighbours. But that's a high position to reach, and few can."

"It's very easy in my opinion not to judge other people. But when life
demands you to judge, then the trouble begins."

"When our own interest comes in, we often make a mess of it and judge
wrong," admitted Mr. Chaffe. "And what I always say to anybody in a fix is
this: to get outside the question and think how it would be if it was all
happening to somebody else. If you've got the sense to do that, you'll often
be surprised to find the light will shine. And you'll often be surprised, also,
to find how much smaller the thing bulks, if you can wriggle out of it
yourself and take a bird's-eye view."

"I expect that's true."

"Oh yes, it's true. I've proved it. A thing happens and you're chin deep in
it. Then you say to yourself, 'Suppose I was dead and looking down on this
job from my heavenly mansion, how would it seem then?' And if you've got
the intellects to do it, then you often get a gleam of sense that you never
will while you're up against the facts and part of 'em. It's like the judge
trying a case, without having any interest in it beyond the will that right
shall be done."

"Men haven't the gift for that."

"They have not; yet even to try to do it stills passion and breeds patience
and helps religion."

"Very good advice, no doubt."

"This coffin will go along early to-morrow morning, and I'll bring half
the hurdles this week in two or three loads; and tell Joe the price be up a
thought since last year. He knows that as well as I do."

Maynard noted the instructions in a little pocket-book and presently

departed. He took a meal of bread and cheese and cider at the inn hard by,
then set out on an extended round, walked to Widecombe, tramped the
Moors, watched the swaleing fires, that now daily burned upon them, and
did not return home until the hour of milking.
 CHAPTER XI

NEW BRIDGE

On New Bridge, over Dart, stood Dinah with the sun warm upon her
face, while a first butterfly hovered on the golden broom at water's edge.
She had sent a message to Johnny by his sister that she would meet him
here, and now, while she waited, she speculated on the difference between
the beauty of the May day and the ugliness of what she was about to do. But
she had decided at last, and having done so, she could only wonder why it
had taken her so many weeks to reach a decision. To her direct instincts
delay had been a suffering and produced a condition of mental bad health;
but it was not for her own sake that she had delayed, and she knew now that
her hesitation had been no kindness to Johnny, though endured largely out
of affection for him. She was convinced, beyond possibility of doubt, that
her regard could not be called love and she had determined with herself, as
she was bound to do, that to marry under such circumstances would be no
marriage in any seemly interpretation of the contract. She had the
imagination to know, however, that what was beaten ground to her—a way
exploited a thousand times by day and sleepless night—was no such thing
for him. He had said that he would have nothing more to do with her until
she named the day, and he was coming now under expectation of hearing
her do so. Instead he must learn that the day could never be named.

She was full of sorrow, but no fear. Dinah had long discounted the effect
of the thing she was called to do. She did not expect anybody to be patient,
or even reasonable, save her step-father.

Johnny appeared punctually, with his gun on his shoulder. They had not
met for more than a month, but he ignored the past and greeted her with a
kiss. She suffered it and reflected that this was the last time he would ever
kiss her.

"At last," he said. "I've hated this job, Dinah; and you'll never know
how much I hated it; but what could I do?"
 "I don't know, Johnny. You could have wondered a bit more why I held
off perhaps."

"And didn't I wonder? Didn't I puzzle myself daft about it? I don't know
now—such a downright piece as you—I don't know now why you hung
back. It wasn't natural."

"Yes it was—everything's natural that happens. It couldn't happen if it

wasn't natural—old Arthur Chaffe said that once and I remembered it."

"If it was natural, then there was a reason," he answered, "and I'd like to
hear it, Dinah—for curiosity."

"The reason is everything, John. I didn't know the reason myself for a
good bit—the reason why I held away from you; and when I did, I was so
put about that it seemed to alter my whole nature and make me shamed of
being alive."

"That's pretty strong. Better we don't go back then. I'll ask no questions
and forget. We'll begin again by getting married."

"No; the reason you've got to hear, worse luck. The reason why I
behaved so strange was this, John: I'd made a terrible mistake—terrible for
both of us. I thought the love that I had for you, and still have for you, and
always shall, was the love of a woman for the man she's going to wed.
Then, like a cloud, it came over me, denser and denser, that it was not.
Listen—you must listen. I examined into it—give me that credit—I
examined into it with all my senses tingling night and day. I never worked
so hard about anything after I'd got over my first fright. And then I saw I'd
slipped into this, being young and very ignorant about love—much more so
than many girls younger than me; because I never was interested in men in
the way they are. I found that out by talking to girls, and by the things they
said when they knew I was tokened to you. They looked at marriage quite
different from me, and they showed me that love is another thing altogether
seen that way than as I'd seen it. They made me terrible uncomfortable,
because I found they'd got a deep understanding that I had not got about it;
and they laughed at me, when I talked, and said I didn't know what love
meant. And—and—I didn't, Johnny. That's the naked truth."
 He was looking at her with a flushed face.

"Get on—get on to the end of it," he said.

"Be patient. I'm bitter sorry. We was boy and girl for so many years, and
I loved you well enough and always shall; but I don't know nothing about
the sort of love you've got for me. The first I heard about it was from Jane.
She knows. She understands far deeper about what love is than I do. I only
know I haven't got it, and what I thought was it didn't belong to that sort of
love at all. Haven't you seen? Haven't you fretted sometimes—many times
—because I couldn't catch fire same as you, when you touched me and put
your arms round me? Didn't it tell you nothing?"

"How the devil should it? Women are different from men."

"Not they—not if they love proper. But how could you know that—you,
who was never in love before? I don't blame you there; but if you'd only
compare notes with other men."

"Men don't compare notes as you call it about sacred things like love."

"Don't they? Then they're finer than us. Women do. Anyway I found
out, to my cruel cost, I was only half-fledged so far as you were
concerned."

"I see. But you needn't lie about it—not to me. You loved me well
enough, and the right way too. You can't shuffle out of it by pretending any
trash about being different from other girls. You loved me well enough, and
if you'd been on-coming like some creatures, I'd have hated you for it. That
was all right, and you knew what you were doing very well indeed. And
you're lying, I say, because it wasn't women have brought you to this. It was
men. A man rather. Be plain, please, for I won't have no humbug about this.
You've found some blasted man you hanker after and think you like better
than me. And it's not the good part in you that have sunk to any such base
beastliness; it's the bad, wicked part in you—the part I never would have
believed was in you. And I've a right to know who it is. And I will know."
 "Hear me then, Johnny. May God strike me dead on this bridge, this
instant moment, if there's any man in the world I love—or even care for. I
tell you that I've never known love and most likely never shall. 'Tis long
odds it be left out of me altogether. And I can't marry you for that good
reason. I didn't come to it in a hurry. For one of my nature I waited and
waited an amazing time, and for your sake I hoped and hoped I'd see
different, and I tried hard to see different. I thought only for you, and I'm
thinking only for you now. It would have been far easier for me to go on
with it than break. Can't you see that? But afterwards—you're a quick man
and you're a man that gives all, but wants all back again in exchange for all;
and rightly so. But what when you'd found, as find you must, that I'd not
loved you as you thought? Hell—hell—that's what it would have been for
you."

"You can spin words to hide your thoughts. I can't. You're a godless,
lying traitor—and—no—no—I call that back. You don't know what you're
saying. Have some mercy on a man. You're my all, Dinah. There's nothing
else to life but you! Don't turn me down now—it's too late. You must see
it's gone too far. You can't do it; you can't do it. I'm content to let it be as it
is. If you don't love me now, I'll make you love me. I'll—all—I'll give all
and want nothing again! It's cruel—it's awful—no such thing could happen.
I believe you when you say there's not another man. I believe you with all
my heart. And then—then why not me? Why not keep your solemn oath
and promise? If anything be left out of you, let me put it in. But there's
nothing left out—nothing. You're perfect, and the wenches that made you
think you wasn't ban't worthy to black your boots. For Christ's sake don't go
back on me—you can't—it wouldn't be you if you did."

"Don't make it worse than it is, dear John. I'm proud you could care for
me so well; but don't you see, oh, don't you see that I can't act a lie? I can't
do it. Everything tells me not to do it. I'm in a maze, but I know that much. I
must be fair; I must be straight. I don't love you like that. I thought I did,
because I was a fool and didn't know better. It can't be. I'm fixed about it."

For a moment he was quiet. Then he picked up his gun, which he had
rested against the parapet of the bridge. His face was twisted with passion.
Then she heard him cock the gun. For a moment she believed that he meant
to shoot her. She felt absolutely indifferent and was conscious of her own
indifference, for life seemed a poor possession at that moment.

"You can kill me if you like," she said. "I don't want to go on living—
not now."

He cursed her.

"Lying bitch! Death's a damned sight too good for you. May your life be
hell let loose, and may you come to feel what you've made me feel to-day.
And you will, if there's any right and justice in life. And get out of Lower
Town—d'you hear me? Get out of it and go to the devil, and don't let me see
your face, or hear your voice in my parents' home no more."

A market cart came down the hill and trundled towards them, thus
breaking into the scene at its climax. John Bamsey turned his back and
strode down the river bank; Dinah hid her face from the man and woman in
the cart and looked at the river.

But the old couple, jogging to Poundsgate, had not missed the man's
gestures.

The driver winked at his wife.

"Lovers quarrelling!" he said; "and such a fine marnin' too. The twoads
never know their luck."

With heavy heart sat Johnny by the river under great pines and heard the
rosy ring-doves over his head fluttering busily at their nest; while Dinah
leant upon the parapet of the bridge and dropped big tears into the crystal of
Dart beneath her.
 CHAPTER XII

AFTERWARDS

The shock of Orphan Dinah's sudden action fell with severe impact in
some directions, but was discounted among those of wider discernment.
The mother of John had seen it coming; his father had not. In a dozen
homes the incident was debated to Dinah's disadvantage; a few stood up for
her—those who knew her best. In secret certain of John's acquaintance
smiled, and while expressing a sympathy with him, yet felt none, but rather
satisfaction that a man so completely armed at all points, so successful and
superior, should receive his first dose of reality in so potent a shape.

The matter ran up and down on the tongues of those interested. His
mother and sister supported Johnny in this great tribulation, the first with
dignity, the second with virulence, hardly abated when she found herself
more furious than John himself.

For after the first rages and intemperate paroxysms in which Jane
eagerly shared, she fancied Johnny was cooling in his rage; and, such are
the resources of human comedy, that anon her brother actually reproved
Jane for some particularly poignant sentiments on the subject of Dinah. He
had set her a very clear-cut example in the agonised days of his grief; but
presently, to the bewilderment of Jane, who was young and without
experience of disappointment, John began to calm down. He roughly shut
up the girl after some poisonous criticism of Dinah, and a sort of alliance
into which brother and sister had slipped, and into which Jane entered with
full force of love for John and hate for Dinah, threatened to terminate.

Jane lessened nothing of her fervid affection for John, however, and it
remained for another man to explain what seemed to her a mystery. He was
not a very far-seeing, or competent person, but he had reached to the right
understanding of Johnny's present emotion.

With Jerry Withycombe Jane fell in beside a track through the forest,
where he was erecting a woodstack, and since their relations were of the
friendliest and Jane, indeed, began to incline to Jerry, she had no secrets
from him and spoke of her affairs.

"What's come to them I don't know," she said. "Father's plucking up

again, and I can see, though Dinah's trying to get a place and clear out, that
he'll come between and prevent it very likely. Mother's at him behind the
scenes, but God knows what they say to each other when they go to bed.
You'd think Dinah wouldn't have had the face to bide in the house a day
after that wickedness; but there she is—the devil. And John ordered her to
go, too, for he told me he had."

"It's your father," answered Jerry. "My sister was telling about it.
Melindy says that Mr. Bamsey's troubled a lot, and though he knows Dinah
has got to go, he's taking it upon himself to decide about where she shall go
and won't be drove."

"I see through that; mother don't," said Jane. "Father only cares for
Dinah really, and he thinks, in his craft, that very like, given time, things
may calm down and her be forgiven. That's his cowardly view, so as he
shall keep her. But nobody shan't calm down if I can help it. I won't live
with the wretch, and so I tell John. Men ban't like us: they don't feel so
deep. They're poor things in their tempers beside us. A woman can hate a
lot better than a man. Why, even Johnny—you'd never believe it; but you'd
almost think he's cooling a bit if it was possible."

"He is," answered Jerry. "And why not? What the hell's the good of
keeping at boiling point over what can't be helped? Especially if, on second
thoughts, you begin to reckon it can be helped."

"What d'you mean by that?" asked Jane.

"Why, you see John's a very determined sort of customer. He's never
took 'no' for an answer from anybody, and he's got an idea, right or wrong,
that a man's will is stronger than a woman's. I thought so, too, till I got to
know what a rare will you've got. But there it is in a word; not two days
agone I met Johnny, and he said where there was life there was hope."

Jane gasped.
 "That's what be in his head then! That's what made him stop me pretty
sharp when I was telling the truth about her?"

Jerry nodded.

"Very likely it might have been. In fact, he ain't down and out yet—in
his own view, anyway. You see, as John said to Lawrence Maynard, and
Maynard told me, 'If Dinah ain't got no other man in sight, she's what you
may call a free woman still.' And I believe that John be coming round to the
opinion that Dinah may yet live to see she was wrong about him."

Jane stared and her thoughts reeled.

"D'you mean to tell me that a man like my brother could sink to think
again of a girl that had jilted him?" she flamed.

"Don't you turn on me," protested Jerry. "It ain't my fault men are like
that. You know John better than I do. But it wouldn't be contrary to nature if
he did want her still. A man in love will stand untold horrors from a
woman; and though it may make you, looking on, very shamed for him—
still, life's life. And I believe, if John thinks he can get Dinah back, he'll
come down off his perch yet and eat as much dirt as she likes to make him."

"It's a beastly thought—a beastly thought!" cried Jane. "But he shan't—

he never shall have her now if I can prevent it. I'd be a miserable woman if I
had to suffer her for a sister-in-law now."

Jerry saw danger in this attitude.

"I always feel just like you feel," he said, "but for God's love, Jenny,
don't you go poking into it. It's a terrible good example of a job where
everybody had best to mind their own business. You let John do what he's
minded to do. Men in love be parlous items, and if he's still that way,
though wounded, then 'tis like a wild tiger a man have fired at and only
hurt. He's awful dangerous now, I shouldn't wonder; and if he wants her still
and counts to get her, God help anybody who came between. He'd break
your neck if you tried to: that I will swear."
 But Jerry was more perturbed at the vision he had conjured than Jane.
For his information she was able to give facts concerning the other side.

"If that's what John's after, he's only asking for more misery then," she
said. "I hope you're wrong, Jerry, for I should never feel the same to John if
I thought he could sink to it; but anyway he needn't fox himself that she'll
ever go back on it again. That much I'm positive certain. Cunning as she is,
I can be more cunning than her, and I know all her sorrow about it and
pretended straightness and honesty was put on. She weren't sorry, and she
never was straight, and I've sworn before to you and will again, that she's
got somebody else up her sleeve."

"Who then?" asked Jerry Withycombe.

"I can't tell you. Lord knows I've tried hard enough to find out; but I
haven't—not yet. Only time will show. It's a man not worthy to breathe the
same air with John you may be sure. She was too common and low ever to
understand John, and his high way of thinking; and she'd be frightened to
marry such a man, because she knows she'd always have to sing small and
take a second place. She's a mass of vanity under her pretences."

"We all know you don't like her; and more don't I, because you don't,"
answered Jerry. "But if you are positive sure she'll never come round to
Johnny again, it might be truest kindness to tell him so. Only for the Lord's
sake do it clever. You may be wrong, and if there's a chance of that, you'd
do far better to leave it alone."

"I'm not wrong; but all the same I shall leave it alone," said Jane. "What
mother and me want is for her to get out of the house, so as we can breathe
again. It's up to father, and father's going to have a bad time if he stands
against mother."

"Dinah won't stop, whether your father wants for her to or not,"
prophesied Jerry.

But a few evenings after this meeting, the situation was defined for the
benefit of Jane and her mother and, with Dinah out of the way at
Ponsworthy, her foster-father endeavoured to ameliorate the existing strain.
He had confided his difficulties to Arthur Chaffe and been counselled to
speak plainly. Indeed, at his wish, the carpenter joined his circle and
supported him.

Mr. Bamsey tried to conceal the fact that Arthur had come to help him,
for his friend not seldom dropped in to supper; but on this occasion Faith
felt aware of an approaching challenge and was not surprised when, after
the evening meal, her husband led the conversation to Dinah Waycott.

"Arthur's my second self," he said, "and I know he'll lift no objection to

listening, even if he don't see with our eyes."

"You needn't say 'our eyes,' father," replied Jane, quick to respond. "Me
and mother——"

But her mother stopped her. Mrs. Bamsey was all for law and order.

"Listen, and don't talk till you're axed to," she said.

"Give heed to me," began Ben. "There's been growing up a lot of fog
here, and Arthur, the friend that he be, was the first to mark it. He pointed it
out to me, all well inside Christian charity, and what I want to do is to clear
it off this instant moment, now while Orphan Dinah's out of the way. We
stand like this. When she threw over Johnny, because her eyes were opened
and she found she couldn't love him in a way to wed him, John ordered her
out of Lower Town. Well, who shall blame him? 'Tweren't vitty they should
clash, or he should find her here in his parents' home. She was instant for
going, and though you think I withheld her from doing so, that ain't fair to
me."

"You do withhold her, father," said Faith Bamsey quietly.

"No, I do not. I come to the subject of Dinah from a point you can't
grasp. For why? She was left to me by my dead first as a sacred and solemn
trust. Mind, I'm not letting my affection for Dinah darken my reason. I grant
I'm very fond of her, and I grant what she's done haven't shook my feelings,
because, unlike you, mother, I believe she's done right. My heart's bled for
my own—for your great trouble and for John's. Nothing sadder could have
come to shake John's faith, and for a time I was fearful for John. The devil
always knows the appointed hour when a soul's weakest, and, coward that
he is, 'tis in our worst moments, when life goes wrong and hope's slipping
away, that he times his attacks. We all know that; and you remember it,
Jane. For he forgets neither the young nor the old. But John has justified his
up-bringing; and the mother in him is bringing him back to his true self."

"You may think so; but——" began Jane.

She was, however, silenced.

"Hear me, and if you can throw light after, Jane, we'll hear you,"
continued Mr. Bamsey. "I say what I think and believe. My trouble be still
alive for John; but my fear be dead. So that leaves Dinah. Her wish and will
is to be gone. She's seeking a proper and fitting place—neither too low nor
too high. She'd go into service to-morrow—anywhere; but I won't have
that."

"And why for not, father?" asked Mrs. Bamsey; "your first was in
service once."

"That's different," he answered. "You must see it, mother. The situation
is very tender, and you must remember my duty to the dead. Would Jane go
into service?"

"No, I would not," answered Jane; "not for anybody. I'd go on the street
first."

Mr. Chaffe was shocked.

"Do I hear you, Jane?" he asked.

"God forgive you, Jane," said her father; then he proceeded.

"My foster-daughter is a much more delicate and nice question than my

own daughter; and mother, with her sharp understanding, knows it. From no
love for Dinah I say so. She's a sacred trust, and if she was a bad girl,
instead of a good one, still she'd be a sacred trust. I'm not standing here for
my own sake, or for any selfishness. I've long been schooled to know she
was going, as we all hoped, to Johnny. And go she must—for her own sake
—and her own self-respect. And if anybody's fretting about her biding here,
it's Dinah's self. But the work she must go to is the difficulty, and that work
has not yet been found in my opinion. Her future hangs upon it and I must
be head and obeyed in that matter."

"She's turned down such a lot of things," said Jane.

"She has not," replied Mr. Bamsey. "She'd do anything and take
anything to-morrow. She was at me to let her go for barmaid to the Blue
Lion at Totnes. And I said, 'No, Dinah; you shan't go nowhere as barmaid
while I live.' And I say it again, meaning no disrespect to the Blue Lion,
which is a very good licensed house."

"She's of age, and if she was in earnest, she could have gone, whether
you liked it or not," said Jane.

Mr. Bamsey grew a little flushed and regarded his daughter without
affection.

"You would—not Dinah," he answered. "Dinah looks to me as her

father, and she won't do nothing I don't hold with, or take any step contrary
to my view. That's because she's got a righter idea of what a girl owes her
father than you have, Jane."

"And what is your view, father?" asked Mrs. Bamsey.

"You know, mother. I want for Dinah to go into a nice family, where the
people will receive her as one of themselves, and where she'll take her place
and do her proper work and go on with her life in a Christian manner, and
not feel she's sunk in the world, or an outcast, but just doing her right share
of work, and being treated as the child of a man in my position have a right
to expect to be treated."

"You won't find no such place, father," said Jane.