Human Hair Keratin Hydrogels Alleviate Rebleeding after

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Human hair keratin hydrogels alleviate rebleeding after intracerebral hemorrhage in a rat model He ye, Qing Qu, Tiantian Luo, yuhua gong, Zongkun Hou, Jia Deng, Yingqian Xu, Bochu Wang, and Shilei Hao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01609 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 23, 2019

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ACS Biomaterials Science & Engineering

Human hair keratin hydrogels alleviate rebleeding after intracerebral hemorrhage in a rat model Ye He a, 1, Qing Qu a, 1, Tiantian Luo a, 1, Yuhua Gong a, Zongkun Hou a, Jia Deng b, Yingqian Xu a, c *, Bochu Wang a, *, Shilei Hao a, *

a

Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of

Bioengineering, Chongqing University, Chongqing 400030, China. b

College of Environment and Resources, Chongqing Technology and Business University,

Chongqing, 400067, China c

Chongqing Engineering Research Center of Pharmaceutical Sciences, Chongqing Medical and

Pharmaceutical College, Chongqing, 401331, China.

∗ Corresponding authors. Tel.:+86 23 6512 0021; Fax: +86 23 6512 0021. E-mail

address:

[email protected]

(Y.

Xu);

[email protected]

(B.

Wang);

[email protected] (S. Hao) 1

These authors contributed equally to this work.

Abstract Surgery is an important therapeutic strategy for intracerebral hemorrhage (ICH) in the clinic and is theoretically beneficial for the outcome of ICH by decreasing hematoma, reducing nervous tissue damage and removing harmful chemicals. However, the outcome of ICH surgery is always unsatisfactory due to postoperative rebleeding. We hypothesized that the injection of hemostatic agents in situ after aspiration surgery could immediately activate hemostasis once rebleeding occurs. Therefore, keratin hydrogels (K-gels) were easily prepared as a hemostatic material via a rehydration method and had a porous structure. Collagenase was injected into the basal lamina to mimic ICH rebleeding, and the K-gels were then injected into the same injured site after 1

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2 h for hemostatic therapy. The hematoma volume was significantly reduced by K-gel treatment, indicating that in situ infusion of the K-gels inhibited hematoma enlargement when rebleeding occurred. Moreover, brain damage, including cell apoptosis, neuroinflammatory reactions and neurological deficits, was also relieved after K-gel treatment. These results suggested that in situ injection of the K-gels into the hematoma area after ICH surgery improves the therapeutic outcome by stopping postoperative rebleeding. K-gels have great potential for clinical hemostatic application due to their excellent hemostatic properties and biocompatibility. Keywords: intracerebral hemorrhage, rebleeding, surgery, keratin, hemostasis

1.

Introduction Intracerebral hemorrhage (ICH) is the second most frequently observed subtype

of stroke after ischemic stroke, 1 which affects 4 million patients worldwide each year, and the average fatality rate at 1 month is approximately 40%. 2 Current strategies for ICH treatment include surgery and conservative treatment in the clinic. However, therapeutic outcomes for ICH are not favorable. 3 However, surgical treatment has some advantages, including decreasing the hematoma volume, reducing nervous tissue damage, relieving local ischemia and removing noxious chemicals.

2, 4-5

Some results

have confirmed that early surgery does not decrease the rate of death or disability at 6 months.

2, 6

Several factors may result in poor outcomes after ICH surgery, including

postoperative rebleeding, low clearance rate, and brain injury induced by surgery.

7

Recently, more surgical techniques for hematoma decompression have been applied for different types of ICH. 8 Specifically, minimally invasive hematoma drainage assisted by tissue plasminogen activator infusion provides a safer method to treat ICH compared to the standard technique. 9 Unfortunately, therapy for rebleeding after surgery remains limited. Hematoma volume is the most significant determinant of outcomes in ICH.

10

Rebleeding commonly occurs in patients who undergo surgery within 4 h (40%), and a relationship between rebleeding and mortality in a 4-hour surgery group has been shown (p=0.030).

11

Monitoring of postoperative rebleeding can be performed using 2


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computed tomography (CT) and magnetic resonance imaging (MRI). 9 Postoperative hematoma is often attributed to pitfalls of surgical techniques, administration of antiplatelet agents, incomplete clot removal, high blood pressure, etc.

12-13

Intensive

blood pressure-lowering treatment is likely to prevent postoperative hematoma enlargement. 14 Additionally, acute hemostatic treatment promotes hemostasis at sites of vascular injury to limit hematoma enlargement. 7 Recombinant factor VIIa (rFVIIa) administration may be considered for reversal of anticoagulation in patients with warfarin-associated ICH.

15

However, a phase 3 trial (FAST) did not show any

functional or survival benefits in rFVIIa-treated patients. 16 Therefore, American Heart Association/ American Stroke Association (AHA/ASA) guidelines do not recommend rFVIIa for routine use in restricting hematoma in patients with ICH. 17 Recently, absorbable hemostatic materials have emerged as standard tools in neurosurgery. 18-19 Topical hemostats are used to stop bleeding by causing blood to clot. Sealants can prevent the leakage of nonclotting fluids from tissue or blood, while adhesive gels are capable of conglutinating various tissues or blood vessels.

20

Therefore, hemostatic agents have great potential to inhibit postoperative rebleeding by being injected into an injury site after hematoma evacuation. Hemostatic agents can act immediately once rebleeding occurs. However, both excellent hemostatic ability and biocompatibility are required for brain implants. Keratins, which are derived from human hair, wool, feathers and other hard tissues, are natural biomaterials categorized as intermediate filaments, which are cytoskeletal components of desmosome cellular junctions.

21

The hemostatic application of keratin proteins has attracted distinct

attention in recent years.

22-24

Furthermore, good biocompatibility of keratin has also

been reported in previous studies. A human hair keratin gel has been injected into a rat brain to intervene in iron overload by slowly releasing minocycline hydrochloride, and the keratin gel was shown to completely degrade within 4 weeks. 25 The aim of this study was to enhance the ICH postoperative outcome by injecting keratin hydrogels (K-gels) at the injury site after hematoma aspiration surgery. Keratin was extracted from human hair, and the K-gels were prepared using a rehydration method. The characterization of keratin extracts and hydrogels were performed by an 3



amino acid analyzer, scanning electron microscope (SEM), Fourier infrared (FT-IR) spectrometer, and rotary rheometer. Furthermore, an ICH animal model was established using a collagenase injection method, and the therapeutic effect of K-gels for postoperative rebleeding was also investigated. 2.

Materials and methods

2.1 Extraction of keratin from human hair Human hair was supplied by local barber shops in Chongqing, China. The methods to extract keratin proteins have been described in our previous study 25. Briefly, human hair was washed with 0.5 % SDS (w/v) to remove surface grease and rinsed extensively with water before air-drying at room temperature. The clean dried hair was reduced using 0.5 M TGA at pH 11.0 for 15 h to break cystine bonds in the hair fibers to solubilize keratin. Subsequently, the reduction solution was retained, and additional keratins were extracted with 100 mM Tris base solution for 2 h, followed by a second extraction with deionized (DI) water. After the reaction mixture was filtered and centrifuged at 6 000 rpm for 40 min at 4 °C, the obtained supernatant was dialyzed by an ultrafiltration flat-sheet membrane (FM1501, Filter & Membrane Technology, China) with a low molecular weight ultrafiltration membrane (5 000 Da). The resulting extracts were lyophilized to obtain keratin powder after concentrating 20-30-fold. 2.2 Characterization of human hair keratin extracts 2.2.1 SDS-PAGE analysis The molecular weight of keratin was analyzed using SDS-PAGE. Electrophoretic separations of human keratins were performed on a 10% (w/v) polyacrylamide separating gel and 5% (w/v) polyacrylamide stacking gel system. The keratin extracts were dissolved in ultrapure water, and then 20 μL of protein solution was mixed with 5 μL of 5× loading buffer. Subsequently, the protein was denatured by boiling the blended solution for 10 min with loading buffer. Then, 10 μL denatured solution and 5 μL protein marker were loaded into the gel well. Separation was performed at 80 V for 1 h, followed by 120 V for 2 h. Then, the gels were stained with 0.02% (w/v) Coomassie Brilliant Blue G-250 for 1 h and destained overnight in an ethanol-acetic acid solution with shaking. Finally, the gel image was obtained with an imaging system. 4


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2.2.2 Amino acid analysis Quantitative analysis of amino acids in human keratin extracts was performed by a fully automated amino acid analyzer (L-8800, Hitachi) using a ninhydrin precolumn derivatization method. In general, the keratin samples were hydrolyzed using 6 N HCl in a glass tube, and a phenylthiocarbamyl-amino acid (PTCAA) derivative was prepared using phenyl isothiocyanate. Then, the hydrolyzed amino acid content was measured by reversed-phase HPLC. 2.3 Preparation of the K-gels Human hair K-gels were fabricated at various concentrations (e.g., 30%, 35%, and 40% (w/v)). Lyophilized keratin powders were reconstituted with ultrapure water in a covered glass container at appropriate concentrations. Then, the mixtures were mixed vigorously by vortexing to achieve homogeneous distributions. Finally, the resulting mixtures of keratin were stirred for 12 h in an incubator at 37 °C to crosslink and form the hydrogels. 2.4 Characterization of the K-gels 2.4.1

Morphological observation The morphological structure of the K-gels (30%, w/v) was observed by a scanning

electron microscope (EVOLS 25, FEI, USA). A lyophilized sample was diced and adhered to a conductive stage. SEM observation was carried out at a voltage of 20 kV after spraying gold for 30 s under vacuum. 2.4.2

FT-IR analysis The chemical structures of the human hair keratin extracts and K-gels were

analyzed using a Fourier transform infrared spectrometer (Nicolet 550, USA).

26

Samples were mixed with potassium bromide at a ratio of 1:100 and pressed into disks. The analysis was conducted in a wavenumber range of 400-4 000 cm−1. 2.4.3

Porosity analysis The porosity of freeze-dried stents of K-gels with different keratin concentrations

was determined using a liquid substitution method. A stent was immersed in a graduated cylinder of a certain volume (V1) of ethanol, and the volume of the liquid containing the gel (V2) was recorded after 30 min. Next, the gel impregnated with 5



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ethanol was removed, and the volume of remaining liquid (V3) was measured. The porosity of the keratin scaffold was calculated by equation (1) as follows:

Porosity  2.4.4

V1  V3  100% V2  V3

(1).

Rheology study The mechanical properties of K-gels with different concentrations (30%, 35%, and

40%, w/v) were investigated by a rotary rheometer (Gemini HR Nano 200, Malvern, USA). The program was initiated in dynamic mode by measuring and recording the elastic modulus (G') and the viscous modulus (G") (n = 3). The rheometer temperature was controlled at 25 °C. The oscillation frequency was set to 0.1-10 Hz, and the fixed strain was 5%. 2.5 Animal experiments 2.5.1

ICH surgery SPF adult male SD rats (260~270 g) were used in this study and were provided by

the Animal Experimental Center of the Army Military Medical University. The feeding and surgical operations of all animals in the experiment were carried out in accordance with an agreement approved by the Animal Use Committee of the Army Military Medical University. The SD rats were weighed and anesthetized with 10% chloral hydrate (0.5 mL/100 g), and the ICH model was established as follows. 25, 27 The scalp was cut approximately 1.5 cm along the midline of the skull. The subcutaneous tissue was separated, and a small hole with a diameter of 1 mm was drilled on the surface of the skull with a miniature skull drill (coordinates: 0.2 mm behind the front ridge and 3.5 mm on the right side). A certain amount of collagenase VII was slowly injected into the right caudate nucleus (depth: 5.5 mm under the bone hole), and the injection was completed at a constant rate within 1 min. Then, the needle was indwelled for 5 min. After the needle was withdrawn, the hole was closed with sterile bone wax, and the wound was sutured in a sterile environment. 2.5.2

Experimental groups The ICH rats were randomly divided into three groups, including the ICH +

vehicle group, the ICH +K-gel group and the sham group for different treatments. 6


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Collagenase was injected to induce hematoma formation, and 10 μL K-gel solution was then injected into the right basal ganglia after 2 h in the ICH + K-gel group. The coordinates of K-gel injection followed those of the ICH model establishment, and brain injection was achieved within 10 min. In addition, 10 μL normal saline was injected into the right basal ganglia in the ICH + vehicle group before establishing the ICH model. Furthermore, for the sham group, the needle was only inserted into the rat brain with the absence of collagenase or the gel, and the other operation remained the same as the other groups. 2.5.3

Hematoma volume measurement

To investigate the effect of K-gels on hemostasis after ICH, different amounts of collagenase (0.2, 0.4 and 0.6 U) were injected into the rat brain to form different volumes of hematomas. ICH rats were perfused with 0.9% saline through the left ventricle, followed by 4% paraformaldehyde (n = 6) after living for 24 h. The brains were removed and immersed in 4% paraformaldehyde for 24 h. Brain tissues were serially sliced into 6- or 8-mm-thick coronal sections at the needle site of the coordinate center. Images of the slices were acquired using a digital camera. Furthermore, the experimental rats from each group were analyzed using a smallanimal magnetic resonance scanner (MAGNETOM Avanto 1.5 T) at 24 h after surgery. The rats were anesthetized with 1.5-2% isoflurane and fixed in the incisors of the coil. During the MRI analysis, the rats were administered 1.5% isoflurane through inhalation to maintain anesthesia. The three-plane reconnaissance imaging sequence was used to adjust the position of the rat head until the central slice was in the plane of the largest bleeding area. The T2-weighted sequence was used for scanning. The scanning field of view was 4 × 4 cm2, and the matrix was 256 × 256. The thickness of the scanning layer was 1 mm. The recovery time was 3 000 ms, and the echo time was 30 ms. MRI image processing was conducted using the publicly available software ImageJ. The area of the hematoma of all brain slices was multiplied by the thickness to calculate the volume of the hemorrhage. 2.5.4

Immunofluorescent Double Labeling Rats were anesthetized (pentobarbital, 60 mg/kg i.p.) and underwent transcardiac 7



perfusion with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4). The brains were removed and placed in 4% paraformaldehyde for 12 h and then immersed in 30% sucrose for 3 to 4 days at 4 °C. Subsequently, the brains were embedded in OCT compound (Sakura Finetek, Inc.) and sectioned on a cryostat (18µm-thick slices). For immunofluorescent double labeling, the primary antibodies were goat anti-NEUN (neuron marker; 1:250 dilution; Chemicon, Temecula, CA) and rabbit anti-GFAP (astrocyte marker; 1:250 dilution; Chemicon, Temecula, CA). Cy3conjugated donkey anti-goat antibody and Alexa Fluor 488-labeled donkey anti-rabbit antibody were used as secondary antibodies. Slides were stained with 4,6-diamidino-2phenylindole (DAPI, Vector Laboratories, Inc., Burlingame, USA) to counterstain the nuclei and mounted with cover slips. 2.5.5

Terminal dUDP nick-end labeling (TUNEL) Staining Quantitative analysis of TUNEL was performed using an ApopTag® Peroxidase

In situ Apoptosis Detection Kit (Sigma-Aldrich, USA). Observation and image acquisition were performed by laser confocal microscopy (Leica TCS SP5). 2.5.6

Behavioral Tests Neurobehavioral tests were performed on the 1st, 3rd, 14th, 28th, 42nd and 56th

days before and after the experiment. For the forelimb placement test, the beard of the affected side of the rat was touched, and the activity of the contralateral upper limb was observed. When the rat responded to whisker irritation, the contralateral forelimb was lifted and placed on the edge of a table to count 1 point. The test was repeated 10 times. When the right basal ganglia of the brain were damaged, the animal exhibited symptoms of weakness on the left front foot. The percentage of the left forelimb successfully lifted onto the table was calculated and compared. For the corner turn test, the rats were placed on a 30° angled device to observe turning left or right, and the test was repeated 12 times. Each interval was no less than 30 s. When the right basal ganglia of the brain were damaged, the rat tended to turn right; therefore, the percentage of turning right was calculated and compared. 2.6 Biocompatibility of the K-gels Rats were anesthetized with chloral hydrate (100 mg/kg, i.p.) and placed in a 8


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stereotaxic frame. Fifty microliters of K-gel solution (30%) was injected into the right basal ganglia, as mentioned for the procedure of ICH surgery. The rats were euthanized, and the brains were stained with hematoxylin and eosin (H&E) at appropriate timepoints (7, 14, 21, and 28 days). In addition, the inflammatory cytokines interleukin1 beta (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the sera of rats were determined with ELISA kits (Neobioscience Technology Co., Ltd., China). 2.7 Statistical Analysis All measurement data are presented as the mean ± SD. ImageJ and LASAF Express View were used for image processing. SPSS 17.0 was used for data analysis, and one-way ANOVA was used to analyze significant differences among the groups. Statistical significance was set at P < 0.05. 3.

Results

3.1 Extraction and characterization of keratin In this study, we used a reduction method to prepare soluble keratin. Briefly, disulfide bonds in proteins were selectively opened with a suitable reducing agent but without destroying the peptide bonds. Notably, TGA was used as the reducing agent and was slightly modified based on the previous extraction method. The extraction solution was purified by isoelectric precipitation and flat ultrafiltration. Therefore, it not only improved the purity of keratin but also shortened the extraction time and greatly improved the extraction process. The percentage of average extraction yield reached 35.81 ± 0.78% (n = 3) based on the dry weight of the hair. The molecular weight of human hair keratin was analyzed by SDS-PAGE. As shown in Fig. 1A, two distinct bands were observed at 45 and 55 kDa as alpha-helical fragments, which were referred to as type I and type II keratins, respectively. This result was consistent with the characteristics of human hair keratin reported in the literature. 23

Type I and type II keratins can spontaneously form hydrogels by crosslinking with

intrinsic cysteine residues. Moreover, some weaker bands were observed at 25 and 35 kDa, suggesting that there was also a very small amount of γ-keratin. These results indicated that the modified extraction method in this study can effectively break the disulfide bond and ensure the integrity of the main chain structure of keratin. 9



The amino acid composition and content of keratin extracts were analyzed by amino acid analysis, and the results were in agreement with those reported in the literature. 23 Seventeen amino acids were identified, with a total content of 74.55 ± 3.52% of the angular protein (n = 3) (Fig. 1B). The results demonstrated that glutamic acid (Glu), cysteine (Cys) and serine (Ser) were the amino acids with the highest levels within the keratin extracts, accounting for 9.34 ± 0.96%, 7.15 ± 0.92% and 6.83 ± 0.66% of the keratin extracts, respectively. 3.2 Preparation and characterization of the K-gels 3.2.1

SEM observation The formation of the K-gels was evaluated by a vial inversion test. The hydrogels

were easily formed at a concentration of 30% (Fig. 2A). Scanning electron microscopy is the most direct method for characterizing gel microstructure. As shown in Fig. 2B, the K-gels exhibited a porous network structure that could provide a pathway for the ingress and egress of water, thereby greatly increasing the water absorption rate of the gels as a rapid hemostatic material (Fig. S1). Different concentrations of the K-gels had a similar porous network structure.

3.2.2

FT-IR analysis The vibrational peak of -SH (2400 cm-1) was weakened in K-gels compared with

the human hair keratin extracts, whereas the characteristic absorption peak of the S-S bond (590 cm-1) was slightly enhanced (Fig. 2C). However, other chemical structures of the K-gels were nearly identical to the keratin extracts, which indicated that only sulfhydryl crosslinking formed a disulfide bond after keratin gelation and that the other structures did not change. 3.2.3

Porosity analysis The porosity of the K-gels gradually decreased with increasing keratin

concentration in a concentration-dependent manner (Fig. 2D). The porosity of the freeze-dried keratin scaffolds with different concentrations of keratin (30 %, 35 %, and 40 %) was 82.14 ± 2.32%, 77.59 ± 2.51% and 68.63 ± 1.74% (n = 3), respectively. 3.2.4

Rheology study 10


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Hydrogels that are used for in situ implantation in the brain to treat rebleeding after ICH should have good mechanical properties and injectability. Therefore, a rheometer was used to analyze the viscoelastic properties of the K-gels with different concentrations. The gel had an obvious concentration dependence (Fig. 2E). The values of the elastic modulus (G') and the viscous modulus (G") were proportional to the concentration. The hydrogel prepared with 30% keratin had a low degree of crosslinking, and G'' was greater than G' in the test frequency range of 0.1-5 Hz. This result indicated a low elasticity and a viscous fluid. The G' of the K-gels with 35% and 40% keratin was much greater than their viscous modulus (G'') over the entire test frequency range. However, the K-gels with 40% keratin had the highest modulus of elasticity, which conformed to the properties of an elastic solid. The injectability was poor, and the porosity was low; therefore, this concentration of the K-gels was not used in the following experiments. 3.3 Animal experiments 3.3.1

Establishment of an ICH animal model First, collagenase was injected to simulate clinical postoperative rebleeding, and

the K-gels were then injected in situ after 2 h. The injection process was achieved within 10 min. Generally, the hematoma volume of the cerebral hemorrhage model was positively correlated with the drug dose. Therefore, different levels of cerebral hemorrhage were induced by injecting a gradient collagenase dose (0.2, 0.4 and 0.6 U) to study the hemostatic effect of the K-gels. The results showed that collagenase successfully induced a brain parenchymal hemorrhage in SD rats. The hematoma volume was proportional to the dose of collagenase. 3.3.2

Hematoma Volume As shown in Fig. 3, different volumes of hematomas were observed by infusing

different doses of collagenase (0.2, 0.4 and 0.6 U). K-gel treatments reduced the hematoma volume, indicating that in situ injection of K-gels inhibited hematoma enlargement when rebleeding occurred. Furthermore, the hematoma volume in the brain for each group was analyzed by MRI. The area of the hematoma per layer was multiplied by the thickness and 11



superimposed to calculate the amount of bleeding. 27 The vehicle groups were injected with different doses of collagenase to form lesions of different sizes (Fig. 4A), which showed a positive correlation. Additionally, the hematoma volume in the lesion area induced by any dose of collagenase was significantly reduced after 10 μL K-gel treatment compared to the vehicle group. ImageJ statistics show the mean hematoma volume induced by different doses of collagenase (Fig. 4B). The hematoma volume of the vehicle group was 122.09 ± 25.25, 170.46 ± 25.25 and 231.86 ± 32.28 mm3 after injecting different doses of collagenase (0.2, 0.4 and 0.6 U, respectively), while the corresponding hematoma volume decreased to 23.05 ± 9.67, 42.09 ± 7.81 and 60.87 ± 16.43 mm3, respectively, after the K-gel treatments. The data confirmed that K-gels had an apparent hemostatic effect on different degrees of cerebral hemorrhaging and could significantly reduce the amount of bleeding (P < 0.001). 3.3.3

Immunofluorescent double labeling analysis Immunofluorescent double-labeled images and statistical results are shown in Fig.

5. The images show a small amount of astrocytes in the sham group and the K-gel group. The cell body was swollen, and reactive hyperplasia occurred, but there was no significant difference in cell density between the sham group (6.48 ± 1.34%) and the K-gel group (6.73 ± 0.41%). However, a large number of astrocytes around the hematoma showed an increase in length and cell surface bulging in the vehicle group (17.13 ± 0.22%), and reactive hyperplasia was more pronounced (P < 0.001). Treatment with the K-gels effectively inhibited the activation of astrocytes and the occurrence of neuroinflammatory reactions. Moreover, axons and dendrites of normal caudate nucleus neurons were observed to be relatively short and regularly and densely arranged. In an ischemic or hypoxic environment, nerve cells die due to the inability to withstand this malignant stimulus. The data emphasized that almost all nerve cells in the vehicle group were necrotic, but the K-gels had a neuroprotective effect and significantly reduced the loss of neurons in the brain (P < 0.001) (sham group: 7.68 ± 2.15%, K-gel group: 5.64 ± 0.44%, vehicle group: 0.63 ± 0.05%). 12


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3.3.4

Analysis of apoptosis Confocal imaging and statistical results are shown in Fig. 6. No obvious TUNEL-

positive staining was observed in the sham group, and the number of apoptotic cells only accounted for 6.53 ± 0.67% of the total cells in the region. However, the number of apoptotic cells significantly increased to 54.31 ± 11.29% in the vehicle group. In contrast, the apoptotic rate of the perihematomal tissue in the K-gel group (22.73 ± 7.48%) was significantly less than that of the vehicle group. This result indicated that the K-gels could not only effectively treat rebleeding after ICH but also significantly reduce neuronal cell damage and apoptosis. 3.3.5

Neurological deficit assessments Limb dyskinesia was assessed by a forelimb uplift test (Fig. 7A) and a corner turn

test (Fig. 7B). The results showed that the neurological function of the rats after ICH was seriously damaged, and contralateral limb dyskinesia occurred. In the vehicle group, the right brains of the rats were severely injured, and the nerve function hardly recovered with time. However, the K-gel group significantly decreased the degree of nerve damage (P < 0.01), and dyskinesia of the rats gradually improved over time. There was almost no difference in neurological deficit between the K-gel group and the sham group after 6 weeks. The results indicated that the K-gels effectively stopped rebleeding after ICH and inhibited hematoma expansion to greatly reduce the neurological deficit caused by hemorrhagic lesions. 3.4 In vivo biocompatibility H&E staining of the brain after K-gel infusion was performed to investigate the biocompatibility of the K-gels. As shown in Fig. 8A, the volume of the K-gels decreased over time, and all K-gels were nearly degraded in the brain within 28 days. In addition, the proinflammatory responses to the feather K-gels were assessed by analyzing the serum levels of inflammatory cytokines. No significant elevations in cytokine levels were observed compared to the sham group (Fig. 8B). The results suggested that the K-gels did not induce adverse tissue inflammation or immunotoxicity after brain infusion. 4.

Discussion 13



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Hematoma volume is considered to be associated with outcome in patients with ICH, and ultraearly hematoma growth can predict poor outcome after acute ICH. 28 Up to 40% of a hematoma grows in the first few hours after ICH onset, 16 which is related to considerable mortality and morbidity. Similarly, postoperative hematoma enlargement (i.e., rebleeding) can also severely influence the treatment outcome of surgery. Although the role of surgery for most patients with spontaneous ICH remains controversial, hematoma evacuation theoretically can prevent herniation, reduce ICP, decrease the mass effect, and stop a secondary injury caused by blood breakdown. 17 In addition, surgery is necessary for patients with a large hematoma volume that produces a mass effect and impairs consciousness. 29 Therefore, once rebleeding occurs, injecting K-gels into the injured site after ICH hematoma aspiration surgery can be used as a hemostatic therapy and improve the outcome of ICH surgery. The hemostatic ability of keratins has been investigated in our previous studies. Keratins obtained from human hair and chicken feathers significantly reduced blood loss and coagulation time in a liver puncture and tail amputation in rat models.

22-23

Furthermore, the hemostatic ability of keratins was evaluated in a porcine lethal extremity hemorrhage model, and keratins significantly increased animal survival and mean arterial pressure compared with chitosan. Therefore, the hemostatic ability of Kgels was not assessed in this study, but the therapeutic effect of K-gels in the treatment of ICH rebleeding was evaluated. The hematoma volume in the brain tissue of each group was analyzed by MRI. The K-gels significantly reduced the amount of bleeding induced by collagenase injection. The ICH animal model is very important for translational hemorrhagic stroke research. Two types of rodent ICH models have been established by autologous whole blood infusion and collagenase infusion methods and have been widely used for preclinical ICH studies. 27 The autologous blood infusion method can precisely control the volume of blood in the brain tissue and has been used for investigating the iron overload mechanism and intervention following ICH. 25, 31 However, the blood infusion method reproduces spontaneous bleeding; therefore, this animal model is not appropriate for ongoing bleeding investigations or bleeding interventions. 27 Therefore, 14


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the collagenase infusion method was used to establish the ICH animal model in the present study. This animal model was reported by Rosenberg in 1990. 32 Normal saline (2 μL) containing 0.01-1 U collagenase was infused over 9 min. Bleeding was observed at 10 min, and erythrocytes were observed around large caudate blood vessels, which indicated that collagenase began to destroy the blood vessels within 10 min. Moreover, extensive bleeding was observed at 4 h after infusion, and the hematoma volume significantly increased within 4 h. 27 Therefore, the timepoint of K-gel infusion was 2 h after ICH model establishment. Additionally, the brain injection of K-gels after 2 h of collagenase infusion avoided collagenase absorption by the K-gel network. K-gels loaded with minocycline hydrochloride have been prepared to reduce iron overload to improve postoperative functional recovery after ICH aspiration surgery. 25 The K-gels were injected into the core of the hematoma after aspiration surgery. A needle was inserted four times to mimic postoperative iron overload, which severely influenced the survival of ICH rats. Therefore, the needle was inserted twice to evaluate the hemostatic effect of the K-gels in this study, which eliminated the influence of surgical frequency on animal survival to a certain degree. The survival ratio of the ICH rats significantly increased after K-gel treatment. A large amount of bleeding was produced after infusion of 0.6 U collagenase, and the hematoma volume reached 231.86 ± 32.28 mm3. The survival ratio of the rats within 24 h was only 17.6% in the vehicle group. In contrast, the survival ration of the rats in the K-gel group increased to 75%. Inappropriate mechanical properties of brain sealants also result in neural injury. Rheology analysis showed that the K-gels with a concentration of 35% keratin were suitable for intracranial implantation. They had a distinct elastic solid character and a high modulus of elasticity. The mechanical behavior of the K-gels was similar to the mechanical properties of adult brain tissue. 33 Moreover, the K-gels with 35% keratin had good injectability and high porosity. Therefore, they were used for the in vivo hemostatic studies. 5.

Conclusions Postoperative rebleeding is a severe problem in the treatment of ICH and is

considered one of the most important factors influencing the outcome of ICH surgical 15



Page 16 of 29

treatment. Therefore, K-gels were developed and injected into the hematoma area after aspiration surgery in the present study. Hemostasis was immediately activated once rebleeding occurred. The K-gels easily formed and had a porous structure, and their mechanical behavior was similar to the mechanical properties of adult brain tissue. Furthermore, the K-gels not only significantly reduced the amount of bleeding induced by collagenase injection but also relieved brain injuries, including cell apoptosis, neuroinflammatory reactions and neurological deficits. The results demonstrated that K-gels are an ideal brain sealant for hemostatic therapy after ICH surgery due to their excellent hemostatic ability and biocompatibility and have great potential to improve ICH postoperative outcomes in the clinic. Acknowledgements The authors acknowledge the financial assistance provided by the National Natural Science Foundation of China [Grant No. 31600770], Chongqing Research Program of Basic Research and Frontier Technology [Grant No. cstc2018jcyjAX0836], Fundamental

Research

Funds

for

the

Central

Universities

[Grant

Nos.

106112017CDJXY230006 and 106112018CDQYSG0007], and the Visiting Scholar Foundation of the Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education [Grant No. CQKLBST-2017-007]. Supporting Information The water absorption rate of K-gels within 120 s. Notes The authors declare no competing financial interests.

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19



Figure Legends Fig. 1. Electrophoretic separation (A) and amino acid composition (B) of keratin. Fig. 2. Images of the vial inversion tests of the keratin solution before and after gel formation (A). SEM images of a K-gel (35%) with magnifications of 170× (B) and 705× (C). FT-IR spectra of keratin and K-gels (D). The percent porosity (E) and mechanical properties (F) of K-gels with different concentrations. Fig. 3. Tissue slices of the sham-, collagenase- and K-gel-treated groups. Fig. 4. MR images at the maximum level of hematoma diameter in the vehicle- and Kgel-treated groups at 24 h post ICH (A). The hematoma volume in vehicle- and Kgel-treated groups after injection of different units of collagenase. Data are presented as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001). Fig. 5. (A) Representative fluorescence images immunostained with GFAP in green, NeuN in red and DAPI in blue (Scale bar: 75 μm). (B) The relative area of GFAP and NeuN in different groups. Data are presented as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001). Fig. 6. TUNEL staining (A) and quantification (B) of perihematomal and cortex apoptotic cells at day 3 post ICH in different groups. Data are presented as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. the sham group, #P < 0.05 and ##P < 0.01 vs. the vehicle group). Fig. 7. Forelimb placement (A) and corner turn (B) scores of sham, vehicle and K-gel groups at 1, 3, 14, 28, 42 and 56 days. Data are presented as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. the sham group, #P < 0.05 and ##P < 0.01 vs. the vehicle group). Fig. 8. (A) H&E staining of a K-gel in the brain at different timepoints after intracerebral infusion. Serum levels of the proinflammatory cytokines IL-1β (B), IL-6 (C) and TNF-α (D) in the sham and K-gel groups of rats at different timepoints after intracerebral infusion.

20


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Table of Contents graphic



Fig. 1. Electrophoretic separation (A) and amino acid composition (B) of keratin.


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Fig. 2. Photographs of the vial inversion tests of the keratin solution before and after gel formation (A). SEM images of K-gel (35 %) with magnification 170 X (B) and 705 X (C). FT-IR spectra of keratin and K-gel (D). The percent porosity (E) and mechanical properties (F) of K-gel with different concentrations.



Fig. 3. The tissue slices of sham, collagenase and K-gel treated groups. 198x137mm (300 x 300 DPI)


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Fig. 4. MR images at the level of maximum hematoma diameter in vehicle and K-gel treated groups at 24 h post ICH (A). The volume of hematoma in vehicle and K-gel treated groups after injection different unit of collagenase. Data are expressed as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001).



Fig. 5. (A) Representative fluorescence images immunostained with GFAP in green, NeuN in red and DAPI in blue (Scale bar: 75 μm). (B) Relative area of GFAP and NeuN in different groups. Data are expressed as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001). 166x83mm (300 x 300 DPI)


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Fig. 6. TUNEL staining (A) and quantification (B) of apoptotic cells at perihematomal and cortex at day 3 post ICH in different groups. Data are expressed as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001 vs sham group, #P < 0.05, ##P < 0.01 vs vehicle group)



Fig. 7. Forelimb placing (A) and corner turn (B) score of sham, vehicle and K-gel groups at 1, 3, 14, 28, 42 and 56 days. Data are expressed as the means ± SD (n = 6) (*P < 0.05, **P < 0.01, and ***P < 0.001 vs sham group, #P < 0.05, ##P < 0.01 vs vehicle group) 174x71mm (300 x 300 DPI)


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Fig. 8. (A) H&E staining of K-gel in brain at different time points after intracerebral infusion. The serum levels of proinflammatory cytokines IL-1β (B), IL-6 (C) and TNF-α (D) in the sham and K-gel groups of rats at different time points after intracerebral infusion. 232x150mm (300 x 300 DPI)


Human Hair Keratin Hydrogels Alleviate Rebleeding after

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