Journal of Refractive Surgery

Translational Science 

Femtosecond Laser Lenticule Transplantation in Rabbit Cornea: Experimental Study

HuiYing Liu, MD; WenQing Zhu, MD; Alice C. Jiang, BS; Alicia J. Sprecher, BS; Xingtao Zhou, MD

Abstract

PURPOSE:

To evaluate the feasibility of femtosecond laser–induced lenticule transplantation in the rabbit cornea and to observe the relative histologic characteristics of corneal tissue and nerve repair after transplantation.

METHODS:

Eight healthy, purebred, New Zealand white rabbits underwent femtosecond laser small-incision lenticule extraction (SMILE) surgery in the right eye. Lenticules were inserted into a femtosecond laser–created corneal stromal pocket in the left eye, which was defined as femtosecond laser corneal lenticule transplantation. Postoperative observation and examination were completed to evaluate the surgery.

RESULTS:

In the early postoperative period, inflammation of the cornea was noted, tissue around the lenticule was edematous, and cells were activated. Tissue edema remained at postoperative day 10. By 1 month, edema had resolved, activated cells gradually became quiescent, and nerve fiber regeneration was observed. By 3 months, the lenticule integrated into the recipient cornea, extracellular matrix gradually cleared, and thicker nerve fibers were noted. By 6 months postoperative, morphology and distribution of the corneal stromal fibers were close to normal, and the number of nerve fibers was reduced.

CONCLUSIONS:

Femtosecond laser corneal lenticule transplantation in rabbits is feasible, as the lenticule was shown to thrive and integrate with the recipient stroma. Nerve regeneration begins after 1 month.

From the Department of Ophthalmology & Vision Science, Eye & ENT Hospital, Fudan University, Shanghai, China (Liu, Zhu, Zhou); and the Department of Surgery, Division of Otolaryngology - Head and Neck Surgery, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin (Jiang, Sprecher).

The authors have no financial or proprietary interest in the materials presented herein.

AUTHOR CONTRIBUTIONS

Study concept and design (H.L., X.Z.); data collection (H.L., W.Z.); analysis and interpretation of data (H.L., W.Z., A.C.J., A.J.S.); drafting of the manuscript (H.L., W.Z., A.J.S.); critical revision of the manuscript (A.C.J., X.Z.); administrative, technical, or material support (A.J.S.)

Correspondence: XingTao Zhou, MD, Dept of Ophthalmology & Vision Science, Eye & ENT Hospital, Fudan University, No. 83 Fenyang Rd, Shanghai, China, 200031. E-mail: jitong0512@163.com

Received: May 12, 2011
Accepted: August 31, 2012

Abstract

PURPOSE:

To evaluate the feasibility of femtosecond laser–induced lenticule transplantation in the rabbit cornea and to observe the relative histologic characteristics of corneal tissue and nerve repair after transplantation.

METHODS:

Eight healthy, purebred, New Zealand white rabbits underwent femtosecond laser small-incision lenticule extraction (SMILE) surgery in the right eye. Lenticules were inserted into a femtosecond laser–created corneal stromal pocket in the left eye, which was defined as femtosecond laser corneal lenticule transplantation. Postoperative observation and examination were completed to evaluate the surgery.

RESULTS:

In the early postoperative period, inflammation of the cornea was noted, tissue around the lenticule was edematous, and cells were activated. Tissue edema remained at postoperative day 10. By 1 month, edema had resolved, activated cells gradually became quiescent, and nerve fiber regeneration was observed. By 3 months, the lenticule integrated into the recipient cornea, extracellular matrix gradually cleared, and thicker nerve fibers were noted. By 6 months postoperative, morphology and distribution of the corneal stromal fibers were close to normal, and the number of nerve fibers was reduced.

CONCLUSIONS:

Femtosecond laser corneal lenticule transplantation in rabbits is feasible, as the lenticule was shown to thrive and integrate with the recipient stroma. Nerve regeneration begins after 1 month.

From the Department of Ophthalmology & Vision Science, Eye & ENT Hospital, Fudan University, Shanghai, China (Liu, Zhu, Zhou); and the Department of Surgery, Division of Otolaryngology - Head and Neck Surgery, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin (Jiang, Sprecher).

The authors have no financial or proprietary interest in the materials presented herein.

AUTHOR CONTRIBUTIONS

Study concept and design (H.L., X.Z.); data collection (H.L., W.Z.); analysis and interpretation of data (H.L., W.Z., A.C.J., A.J.S.); drafting of the manuscript (H.L., W.Z., A.J.S.); critical revision of the manuscript (A.C.J., X.Z.); administrative, technical, or material support (A.J.S.)

Correspondence: XingTao Zhou, MD, Dept of Ophthalmology & Vision Science, Eye & ENT Hospital, Fudan University, No. 83 Fenyang Rd, Shanghai, China, 200031. E-mail: jitong0512@163.com

Received: May 12, 2011
Accepted: August 31, 2012

 

Corneal refractive surgery usually results in corneal thinning. Patients with thinner corneal thickness or higher degrees of myopia generally face more risk than others.1 A newer alternative technique for myopic patients, small-incision lenticule extraction (SMILE), results in corneal thickening.

Small-incision lenticule extraction2,3 is performed using a femtosecond laser to carve out an intrastromal lenticule and a small incision. Through this incision, the lenticule is removed while the rest of the cornea remains intact. Femtosecond laser lenticule transplantation has the following potential clinical significance: can be used in myopia, hyperopia, astigmatism, and presbyopia treatment; can improve corneal thickness; can be used for the treatment of corneal stromal opacity and keratoconus; and donor materials can be harvested in vivo, which may ameliorate the present shortage of cornea transplant material.

In this study, SMILE surgery was performed on one eye of an animal and a corneal flap was created in the fellow eye using a femtosecond laser. Only the edge above the quadrant was separated and a limited-sized incision was created. Therefore, an intrastromal pocket was created. The lenticule extracted from the first eye was inserted into the corneal pocket that was created in the fellow eye. This insertion procedure was named femtosecond laser corneal lenticule transplantation. Recovery of the corneal nerve and tissue was observed and reported.

Materials and Methods

Eight healthy, purebred, adult New Zealand white rabbits (EENT Hospital, Fudan University) weighing 2.5 to 3.5 kg without obvious anterior segment lesions evaluated by slit-lamp microscopy were used in this study.

Rabbits underwent SMILE surgery on the right eye with a corneal lap diameter of 7.0 mm, thickness of 100 μm, and side-cut angle of 90°. The diameter of the lenticule was 6.0 mm, minimum thickness was 15 μm, side-cut angle was 90°, and refractive correction sphere was −4.00 diopters (D). Femtosecond laser corneal lenticule transplantation was performed on the left eye. A corneal flap with a diameter of 7.0 mm, thickness of 100 μm, and 90° side-cut angle was created using a femtosecond laser. The edge of the above quadrant was separated and an incision, limited to as short as possible, was made into an intrastromal pocket. The lenticule extracted from the first eye was inserted into the intrastromal pocket in the fellow eye with an iris repositor and smooth microforceps. The VisuMax femtosecond laser system (Carl Zeiss Meditec, Jena, Germany) was used for both procedures. Figure 1 illustrates the surgical procedure. Tobramycin and dexamethasone eye ointment was applied immediately after surgery and three times per day thereafter for the first week.

Surgical procedure. R-1) Femtosecond laser ablation, with the bottom layer completed, and the upper layer still under operation. R-2) Dissection of the upper layer. R-3) Dissection of the bottom layer. R-4) Extraction of the stromal lenticule. L-1) Femtosecond laser ablation. L-2) Dissection of the pocket. L-3) Insertion of the lenticule (arrow). L-4) Adjustment of the lenticule (arrow) position. L-5) Flattening of the flap and lenticule. L-6) Successfully transplanted state. R = right eye, L = left eye

Figure 1. Surgical procedure. R-1) Femtosecond laser ablation, with the bottom layer completed, and the upper layer still under operation. R-2) Dissection of the upper layer. R-3) Dissection of the bottom layer. R-4) Extraction of the stromal lenticule. L-1) Femtosecond laser ablation. L-2) Dissection of the pocket. L-3) Insertion of the lenticule (arrow). L-4) Adjustment of the lenticule (arrow) position. L-5) Flattening of the flap and lenticule. L-6) Successfully transplanted state. R = right eye, L = left eye

Observation and examination was performed on postoperative days 1, 10, and 20 and at 1, 3, and 6 months. One animal was examined at each point, first by slit-lamp microscopy and confocal microscopy in vivo. The animal was euthanized and the cornea extracted. The corneal material was separated; one half underwent hematoxylin-eosin (HE) staining for light microscopy examination and the other half was examined with transmission electron microscopy.

Results

Figure 2 shows the cornea at all postoperative examinations.

Examination results. Slit-lamp microscopy (SLM) -1D) Central area of cornea shows mild edema, arrow 1 points to the incision, edema was obvious around the incision, and arrow 2 points to the discernible edge of the inserted lenticule. SLM-10D) Central area of cornea shows slight edema, incision was well positioned. SLM-6M) Cornea was clear. Hematoxylin-eosin–light microscopy (HE-LM)-1D) Arrow 1 shows the lenticule inserted, arrow 2 shows edema. HE-LM-10D) Arrow 3 shows the lenticule, area around the lenticule was still edematous and fibers were corrugated. HE-LM-1M) Edema absorbed, the lenticule can still be distinguished from the change of direction in collagen arrangement. HE-LM-3M) The lenticule was merged, the fibers were arranged regularly and tightly. HE-LM-6M) Morphology and distribution of the corneal stromal fibers was close to normal. Confocal microscopy (CMTF)-1D) Hyper-reflective spots represent inflammatory cells, bright reflective area indicates edema, and corneal tissue was indistinct. CMTF-10D) Bright reflective area of corneal edema, no nerve fibers were seen. CMTF-1M) Hyper-reflective spots and bright reflective area were seldom seen, arrows point to the nerve fibers, tiny and short. CMTF-3M) Corneal tissue was clear, arrow points to the nerve fibers, long and thick. CMTF-6M) Arrows point to the nerve fibers, not so thick but with more branches. Electron microscopy (EM) -1D) The tissue near the lenticule presents edema, arrow 1 shows the implanted lenticule, cells near the lenticule were observed with light nucleus, extensive rough endoplasmic reticulum (RER) and mitochondrial swelling (which indicate active secretion), no living cells in the lenticule, only cell debris could be seen. EM-10D) The nucleus color was still light, extensive RER indicated active secretion, the fibrils were slightly disordered and corrugated. EM-1M) The fibrils appeared orientated, the fibroblast nucleus was getting dark, no obvious RER extension. EM-3M) The fibrils were arranged regularly, cell nucleus became hyperchromatic with no obvious expansive RER. EM-6M) Fibrils orientation was tight and regular, cell nucleus was hyperchromatic with no expansive RER, showing a quiet resting state. 1D = 1 day, 10D = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months

Figure 2. Examination results. Slit-lamp microscopy (SLM) -1D) Central area of cornea shows mild edema, arrow 1 points to the incision, edema was obvious around the incision, and arrow 2 points to the discernible edge of the inserted lenticule. SLM-10D) Central area of cornea shows slight edema, incision was well positioned. SLM-6M) Cornea was clear. Hematoxylin-eosin–light microscopy (HE-LM)-1D) Arrow 1 shows the lenticule inserted, arrow 2 shows edema. HE-LM-10D) Arrow 3 shows the lenticule, area around the lenticule was still edematous and fibers were corrugated. HE-LM-1M) Edema absorbed, the lenticule can still be distinguished from the change of direction in collagen arrangement. HE-LM-3M) The lenticule was merged, the fibers were arranged regularly and tightly. HE-LM-6M) Morphology and distribution of the corneal stromal fibers was close to normal. Confocal microscopy (CMTF)-1D) Hyper-reflective spots represent inflammatory cells, bright reflective area indicates edema, and corneal tissue was indistinct. CMTF-10D) Bright reflective area of corneal edema, no nerve fibers were seen. CMTF-1M) Hyper-reflective spots and bright reflective area were seldom seen, arrows point to the nerve fibers, tiny and short. CMTF-3M) Corneal tissue was clear, arrow points to the nerve fibers, long and thick. CMTF-6M) Arrows point to the nerve fibers, not so thick but with more branches. Electron microscopy (EM) -1D) The tissue near the lenticule presents edema, arrow 1 shows the implanted lenticule, cells near the lenticule were observed with light nucleus, extensive rough endoplasmic reticulum (RER) and mitochondrial swelling (which indicate active secretion), no living cells in the lenticule, only cell debris could be seen. EM-10D) The nucleus color was still light, extensive RER indicated active secretion, the fibrils were slightly disordered and corrugated. EM-1M) The fibrils appeared orientated, the fibroblast nucleus was getting dark, no obvious RER extension. EM-3M) The fibrils were arranged regularly, cell nucleus became hyperchromatic with no obvious expansive RER. EM-6M) Fibrils orientation was tight and regular, cell nucleus was hyperchromatic with no expansive RER, showing a quiet resting state. 1D = 1 day, 10D = 10 days, 1M = 1 month, 3M = 3 months, 6M = 6 months

Slit-Lamp Microscopy

Mild corneal edema was seen on postoperative day 1, with greater edema noted around the incision. After 10 days, mild corneal edema was observed, and edema around the incision was mostly absorbed and the incision margins were positioned well. At 6 months, the cornea was clear and had returned to normal.

Hematoxylin-Eosin Staining Light Microscopy

Corneal tissue was edematous on the first day, especially in the area near the lenticule. On postoperative day 10, bright edematous areas and corrugated fibers could still be seen. After 1 month, the edema was absorbed; however, the lenticule position could still be distinguished from the change of direction in collagen arrangement. At 3 months, the lenticule had integrated into the recipient cornea and fibers were regularly arranged. At 6 months, the morphology and distribution of the corneal stromal fibers were close to normal.

Confocal Microscopy In Vivo

A large number of hyper-reflective inflammatory cells and blurred stromal nuclei, a bright reflective area of corneal edema, and indistinct corneal tissue were observed on postoperative day 1. A bright reflective area of corneal edema and lightly blurred stromal nuclei could be seen on day 10. No nerve fibers were seen. At 1 month, corneal edema absorbed, stromal nuclei became sharp, and nerve fiber regeneration had begun. At 3 months, the morphology and distribution of the corneal stromal cells were close to normal and nerve distribution was minimal, with regenerate fibers becoming thicker. At 6 months, the cornea showed a normal, quiet state, and nerve fibers became branched but still lacked in number.

Transmission Electron Microscopy

On postoperative day 1, edema was present in the tissue near the lenticule, cells near the lenticule were observed with light nucleus, extensive rough endoplasmic reticulum (RER) and mitochondrial swelling, which indicated active secretion, were observed. No viable cells were noted in the lenticule, only cell debris. On day 10, the fibrils were slightly disordered and corrugated, the nucleus was pale, and extensive RER and mitochondrial swelling persisted. At 1 month, the fibrils appeared orientated, the fibroblast nucleus was getting dark (indicating stability), and no obvious RER extension was noted. At 3 months, the fibrils were arranged regularly and the cell nucleus became hyperchromatic with no obvious expansive RER. At 6 months, the fibril orientation was tight and regular and cell nucleus was hyperchromatic with no expansive RER, showing a quiet resting state.

Complications

In one rabbit, the flap shifted on the first postoperative day due to palpebra tertia trauma. In another animal, the eye became infected after surgery and developed keratoleukoma. These corneas were eliminated from the evaluation.

Discussion

Early efforts in corneal stromal lenticule transplantation with ground frozen homograft corneal lamellae4 and hydrogel material5,6 have been reported, with visual outcomes of 20/507 and 20/40.8,9 Experimental animal studies10 and clinical follow-up11 have not shown satisfactory outcomes. The development of traditional keratophakia was limited by complex operating procedures, undesirable transplantation material, and poor postoperative vision.

In this experimental study, an intrastromal pocket was made between the lamellae with a femtosecond laser and the lenticule harvested by the SMILE procedure was implanted. Results showed that the lenticule was not only viable, but had integrated with the recipient stroma. Creation of the stromal lenticule and pocket by the femtosecond laser is accurate and may avoid a decrease in postoperative corrected visual acuity caused by an uneven surface when using a manual keratome12,13 and tissue loss caused by trephine, achieving a better fit between the donor and recipient cornea.14–18 Because of the absence of sutures in both the donor and host eyes, the inflammatory response or transplant rejection normally evoked by sutures, as well as iatrogenic astigmatism caused by suture positioning and tension, was reduced. Tissue damage was less with the femtosecond laser than a keratome, postoperative reactions were minimized, and recovery was faster.19 Implantation of the donor lenticule within the recipient stroma, relatively insulated from the variety of immune factors concentrated in bodily fluids such as tears and aqueous humor, may reduce the risk of immune factor–induced transplantation rejection.20

In this study, a radian incision of approximately 5 mm was made to create the intrastromal pocket, keeping all other edges intact, achieving the advantages of both small incision2,3 and high accuracy of a femtosecond laser.19 Small-incision lenticule extraction is not only a refractive surgery, but also a surgery that thickens the cornea. With this technique, it may be possible to perform further treatments such as surface ablation, although it is not yet clear if the added lenticule actually increases corneal biomechanical strength.

In this study, the pathologic response of the cornea was obvious in the early period after femtosecond laser lenticule transplantation. Corneal edema was not absorbed until postoperative day 10. Tissue repair response was relatively quiet until 3 months and stabilized by 6 months, showing that clinical application of this technology needs a longer drug support and observation period.

Postoperative nerve regeneration was in accordance with reports after LASIK,21–23 but better than penetrating keratoplasty (PK).24 Nerve damage in both LASIK and PK was breakage, with the only difference being the interface material, which was autologous or allogeneic. In eyes that underwent PK, Chen et al24 found that nerve regeneration appeared earlier in the fresh tissue group than in the glycerin-cryopreserved tissue group. The interface in our study was autologous, and implantation was performed soon after lenticule extraction, which ensured the freshness of the implantation material, which may explain why the nerve regeneration was lighter than PK, close to LASIK.

Centralized positioning of the lenticule is challenging and may result in better refractive improvement. In this study, we tried to limit the experiment condition, as even animals may have different reactions, and we were able to demonstrate the feasibility of femtosecond laser corneal lenticule transplantation. The lenticule can survive and merge with the recipient stroma, and nerve regeneration begins after a period of time. Limitations of our study include the relatively short observation period and limited number of subjects. Future studies should focus on long-term outcomes, centralized positioning, optical effects, and allogeneic transplantation results.

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10.3928/1081597X-20121115-05

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