PENYAKIT SARAF

Paraplegia pada Anjing

Oleh :

Dharma Audia Samsuri

2009611034

Kelompok 17G

LABORATORIUM ILMU PENYAKIT DALAM VETERINER

PENDIDIKAN PROFESI DOKTER HEWAN

FAKULTAS KEDOKTERAN HEWAN

UNIVERSITAS UDAYANA

2021
 PENDAHULUAN

Paraplegia merupakan suatu kondisi hilangnya kemampuan untuk menggerakkan

anggota tubuh yang meliputi kedua tungkai dan organ panggul, yang disebabkan karena
kerusakan syaraf pada tulang belakang. Hal ini menyebabkan inervasi dari otak terputus,
sehingga terjadi kelumpuhan otot anggota gerak bawah. Menurut Thomson & Hans (2012)
Paraplegia dikategorikan dalam dua penyebab yaitu karena trauma atau penyakit.

Pada kasus ini Anjing mengalami paraplegia yang dimulai tiga minggu yang lalu.
Berdasarkan temuan computed tomography (CT), kondisi tersebut didiagnosis sebagai herniasi
intervertebralis Hansen tipe I di ruang intervertebralis T12-T13 (Sharun et al., 2020). Herniasi
intervertebralis (IVDH) adalah salah satu penyebab umum cedera tulang belakang pada anjing.
Hal ini biasanya dirawat dengan melakukan dekompresi bedah yang melibatkan pengangkatan
bahan cakram yang diekstrusi.

REKAM MEDIK

1. Signalement dan Anamnesa

Seekor anjing Beagle jantan berumur empat tahun mengalami paraplegia yang dimulai
tiga minggu yang lalu. Pasien sebelumnya pernah dirawat dan ditemukan refrakter terhadap
penatalaksanaan medis menggunakan steroid dan terapi suportif lainnya.
2. Pemeriksaan Klinis
Pada saat pemeriksaan klinis menunjukkan tanda-tanda anjing tersebut berjalan dengan
menyeret badan panggulnya yang diduga paraplegia. Semua reflex tulang belakang (reflex
patella, reflex fleksor, dan reflex perianal) ditemukan normal dan hewan memiliki reflex
buang air kecil normal.
3. Pemeriksaan Penunjang
Pemeriksaan penunjang yang dilakukan pada kasus ini menggunakan radiografi dan
Computed tomography (CT). Pada pemeriksaan radiografi mengidentifikasi berkurangnya
ruang diskus antara tubuh vertebral T12 dan T13 vertebrae toraks (Gambar 1). Computed
tomography dilakukan untuk melokalisasi lokasi lesi. CT scan mengidentifikasi material
disk hernia di dalam kanal vertebralis dari T12 dan T13 vertebra toraks. Material disk yang
termineralisasi dari ruang diskus intervertebralis T12-T13 ditemukan mengalami herniasi
ke dalam kanal vertebral, mengakibatkan kompresi medula spinalis fokal (Gambar 2-3).
 Gambar 1. Radiografi lateral tulang belakang thoraco-lumbar. Mengurangi ruang
intervertebral antara T12 dan T13 vertebra toraks (Panah kuning).
Sumber : Sharun et al., 2020

Gambar 2. Gambara CT bidang transversal menunjukkan adanya material disk yang

diekstrusi di kanal vertebra T12. (a) Gambaran CT normal untuk perbandingan. (1) Processus
Spinosus (2) Vertebral canal (3) Vertebral body (b) Material disk yang memanjang ke kanal
medulla spinalis. (4) Lamina vertebral (5) spinal cord (6) material yang diekstrusi menekan
spinal cord.
Sumber : Sharun et al., 2020
 Gambar 3. (a) Gambaran CT yang dilakukan secara dorsal dari anjing yang sama
memungkinkan lokalisasi lesi yang akurat pada ruang intervertebralis T12-T13 (panah). (b)
Gambaran CT sagittal menunjukkan material diskus intervertebralis yang terdislokasi yang
terlihat massa hiper-atenuasi di kanal vertebral (panah).
Sumber : Sharun et al., 2020

4. Diagnosis
Menurut hasil dari pemeriksaan klinis dan pemeriksaan penunjang diagnosis kasus ini
sebagai paraplegia sekunder akibar herniasi intervertebralis I Hansen di ruang invertebralis
T12-T13.
5. Prognosis
Prognosis kasus yang terkena herniasi invertebralis (IVDH) dilaporkan sangat
bergantung pada ada atau tidaknya persepsi nyeri dalam (Olby et al., 2003). Alasan
prognosis yang baik dalam kasus ini dapat dikaitkan dengan konservasi persepsi rasa sakit
yang mendalam meskipun persepsi rasa sakit dangkal kurang.
6. Treatment
Pemilik menolak permintaan dekompresi bedah karena sifatnya invasive. Oleh karena
itu, menyarankan terapi sel induk menggunakan BM-MSC sebagai upaya untuk
membalikkan deficit neurologis yang terkait dengan herniasi disk intervertebral Hansen
tipe I. Sumsum tulang diambil dari anjing sehat yang dibawa ke klinik untuk operasi elektif
dengan persetujuan pemilik hewan. Hewan dibius sebelum pengambilan sumsum tulang
(krista iliaka) sesuai protocol yang dijelaskan oleh Sharun et al., (2020).
Untuk penyuntikan sel induk, anjing dibius dengan xylazine (1 mg / kg berat badan)
diikuti ketamin (5 mg / kg berat badan) secara intramuskuler. Suspensi sel yang disiapkan
(1ml) dari BM-MSC alogenik yang mengandung 1 x 106 sel / ml ditransplantasikan secara
perkutan ke parenkim medulla spinalis di lokasi cedera. Hewan-hewan tersebut diposisika n
dalam posisi berbaring sternal dan jarum tulang belakang ukuran 26 dimasukkan pada
antarmuka T12-T13. Setelah ujungnya memasuki parenkim, setengah dari sel induk
disuntikkan secara perlahan ke sumsum tulang belakang. Setengah sisanya disuntikkan
sambil menarik jarum dari parenkim sumsum tulang belakang untuk menyimpan sel di
beberapa lapisan. Sebanyak empat dosis diberikan pada selang waktu 15 hari bersama
dengan terapi suportif yang meliputi methylcobalamin (500mcg / anjing setiap 12 jam) dan
gabapentin (10 mg / kg berat badan setiap 12 jam) secara oral selama periode penelitian.

Gambar 4. Grafik yang terlihat mengalami peningkatan progresif fungsi neurologis.

Peningkatan yang signifikan pada skor teramati setelah injeksi BM-MSC pertama.
Sumber : Sharun et al., 2020

Hasil dari treatment menunjukkan perbaikan yang signifikan terlihat setelah dosis
pertama BM-MSC. Hewan tersebut mulai menahan beban penuh pada tungkai belakangnya
setelah dua dosis. Perbaikan juga diamati di skor neurologis yang menunjukkan
peningkatan fungsi sensorik. Peningkatan yang sangat baik diamati pada persepsi nyeri
(nosisepsi) yang menyebabkan kembalinya persepsi nyeri superfisial.
 Gambar 5. (a) Hari 0 - Paraplegia dengan menyeret tungkai belakang. (b) Hari 30 - Mulai menahan
beban pada tungkai belakang. (c) Hari 45 – penopang beban pada tungkai belakang yang normal,
tetapi mengidap ataksia. (d) Hari 60 – gaya berjalan hampir normal dengan ataksia ringan. (e) Hari 90
– kembalinya perilaku normal (menggaruk telinga dengan tungkai belakang) dan anjing mulai
bergerak naik turun tangga dengan mudah.

PEMBAHASAN

Paraplegia merupakan suatu kondisi hilangnya kemampuan untuk menggerakkan

anggota tubuh yang meliputi kedua tungkai dan organ panggul, yang disebabkan karena
kerusakan syaraf pada tulang belakang. Hal ini menyebabkan inervasi dari otak terputus,
sehingga terjadi kelumpuhan otot anggota gerak bawah. Kerusakan pada medulla spinalis
segment thoraco-lumbo-sacral akan mengakibatkan hilangnya fungsi motorik/sensor ik
Menurut Thomson & Hans (2012) Paraplegia dikategorikan dalam dua penyebab yaitu karena
trauma atau penyakit. Penyebab paraplegia pada umumnya adalah cedera pada sumsum tulang
belakang atau spinal cord injury (SCI), spondylitis tuberculosis, gangguan genetik (hereditary
spastic paraplegia), gangguan yang sifatnya menurun atau congenital, infeksi, penyakit
autoimun, syrinx (gangguan korda spinalis) (Kohnle, 2011).

Pada kasus ini Anjing mengalami paraplegia. Berdasarkan temuan computed

tomography (CT), kondisi tersebut didiagnosis sebagai herniasi intervertebralis Hansen tipe I
di ruang intervertebralis T12-T13 (Sharun et al., 2020). Herniasi intervertebralis (IVDH)
adalah salah satu penyebab umum cedera tulang belakang pada anjing. Terjadi herniasi
intervertebralis (IVDH) paling sering di daerah torakolumbar, menyebabkan defisit neuron
motorik atas (UMN) di tungkai panggul (Ruddle et al., 2006). Anjing yang terkena penyakit
cakram intervertebralis torakolumbal mungkin memiliki gejala klinis klasik mulai dari nyeri
punggung hingga kelumpuhan. Diagnosis IVDH pada anjing dapat dilakukan dengan
menggunakan modalitas diagnostik seperti myelography, computed tomography (CT), dan
magnetic resonance imaging (MRI). Di antaranya, MRI medan tinggi dianggap sebagai
modalitas ideal untuk diagnosis anjing dengan penyakit sumsum tulang belakang.

Dalam kasus ini seekor anjing Beagle jantan berumur empat tahun mengalami
paraplegia yang dimulai tiga minggu yang lalu dengan gejala klinis tersebut berjalan dengan
menyeret badan panggulnya yang diduga paraplegia. Selanjutnya dilakukan pemeriksaan
penunjang dengan radiografi dan CT scan. Pada pemeriksaan radiografi mengidentifikas i
berkurangnya ruang diskus antara tubuh vertebral T12 dan T13 vertebrae toraks. CT scan
mengidentifikasi material disk hernia di dalam kanal vertebralis dari T12 dan T13 vertebra
toraks. Menurut hasil dari pemeriksaan klinis dan pemeriksaan penunjang diagnosis kasus ini
sebagai paraplegia sekunder akibar herniasi intervertebralis I Hansen di ruang invertebralis
T12-T13. Untuk kasus ini treatment yang digunakan yaitu BM-MCS alogenik untuk implantasi
perkutan tanpa melakukan operasi dekompresi.

Transplantasi perkutan dari BM-MSC yang diinduksi secara neurogenik telah

digunakan untuk pengobatan paraplegia pada anjing akibat penyakit diskus intervertebralis
setelah melakukan dekompresi bedah (Besalti et al., 2015). Anjing dengan cedera sumsum
tulang belakang kronis (SCI) sekunder akibat IVDH torakolumbar telah berhasil diobati dengan
sel induk sumsum tulang janin yang disuntikkan intrameduler segera setelah hemilaminektomi
(transplantasi intra-operatif) (Sarmento et al., 2014). Selanjutnya, transplantasi sel induk
mesenkim yang diturunkan dari adiposa ke dalam parenkim sumsum tulang belakang yang
cedera setelah operasi dekompresi dikaitkan dengan hasil klinis yang lebih baik dibandingkan
dengan operasi dekompresi saja (Kim et al., 2016).

Kesimpulannya, transplantasi perkutan aBM-MSCs mungkin telah membantu anjing

mendapatkan kembali fungsi neurologisnya sepenuhnya tanpa melakukan dekompresi bedah.
Pengiriman sel induk perkutan lebih disukai untuk menghindari kebutuhan intervensi bedah.
Oleh karena itu teknik non-invasif ini dapat dievaluasi lebih lanjut untuk kemanjurannya dalam
mengelola paraplegia sekunder untuk Hansen tipe I IVDH.
 DAFTAR PUSTAKA

Besalti, O; Can, P; Akpinar, E; Aktas, Z; Elcin, AE and Elcin, YM (2015). Intraspinal

Transplantation of Autologous Neurogenically-Induced Bone Marrow-Derived
Mesenchymal Stem Cells in the Treatment of Paraplegic Dogs without Deep Pain
Perception Secondary to Intervertebral Disk Disease. Turk. Neurosurg., 25(4): 625–
632.

Kim, Y; Lee, SH; Kim, WH and Kweon, OK (2016). Transplantation of adipose derived
mesenchymal stem cells for acute thoracolumbar disc disease with no deep pain
perception in dogs. J. Vet. Sci., 17(1): 123–126.

Kohnle, D. (2011). Risk factors. Paraplegia, 907, 550–6100.

Olby, N; Levine, J; Harris, T; Muñana, K; Skeen, T and Sharp, N (2003). Long-term functional
outcome of dogs with severe injuries of the thoracolumbar spinal cord: 87 cases
(1996–2001). J. Am. Vet. Med. Assoc., 222(6): 762-769.

Ruddle, TL; Allen, DA; Schertel, ER; Barnhart, MD; Wilson, ER; Lineberger, JA; Klocke,
NW and Lehenbauer, TW (2006). Outcome and prognostic factors in non-
ambulatory Hansen Type I intervertebral disc extrusions: 308 cases. Vet. Comp.
Orthop. Traumatol., 19(1): 29-34.

Sarmento, CA; Rodrigues, MN; Bocabello, RZ; Mess, AM and Miglino, MA (2014). Pilot
study: bone marrow stem cells as a treatment for dogs with chronic spinal cord
injury. Regen. Med. Res., 2(1): 1-9.

Sharun, K; Kumar, R; Chandra, V; Saxena, AC; Pawde, AM; Kinjavdekar, P; Dhama, K;

Amarpal, and Sharma, GT (2020) Percutaneous transplantation of allogenic bone
marrow-derived mesenchymal stem cells for the management of paraplegia
secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Sci., 10: 22099.

Thomson, C. E., & Hahn, C. (2012). Veterinary Neuroanatomy: A Clinical Approach. Elsevier
Ltd.
See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/349684457

Percutaneous transplantation of allogenic bone marrow-derived

mesenchymal stem cells for the management of paraplegia secondary to
Hansen type I intervertebral disc herniation in a...

Article  in  Iranian Journal of Veterinary Research · March 2021

DOI: 10.22099/IJVR.2021.38613.5620

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Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

Scientific Report

Percutaneous transplantation of allogenic bone marrow-derived mesenchymal

stem cells for the management of paraplegia secondary to Hansen type I
intervertebral disc herniation in a Beagle dog

Khan Sharuna*, Rohit Kumara, Vikash Chandrab, Abhishek Chandra Saxenaa, A. M. Pawdea,
Prakash Kinjavdekara, Kuldeep Dhamac, Amarpala*, G. Taru Sharmab

a
Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh
b
Division of Physiology and Climatology, ICAR-Indian Veterinary Research Institute, Izatnagar,
Bareilly, Uttar Pradesh
c
Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh

*Corresponding authors. Tel.: +91 7012649375

E-mail address: sharunkhansk@gmail.com and dramarpal@gmail.com

Acknowledgements

The authors are thankful to Director, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly,
India for providing necessary research facilities to carry out this work.

Funding

Authors received no funding/grant support for this study.

Ethics approval

Written consent was obtained from the owner before performing the therapy. All procedures performed
are in accordance with the ethical standards of the institution at which the studies were conducted.

1|Page
Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

Abstract
Background: Intervertebral disc herniation (IVDH) is one of the common causes of spinal cord injury
in dogs. It is commonly treated by performing surgical decompression that involves the removal of
extruded disc material. However, the recovery rates after surgical interventions are variable and many
times unsatisfactory. This report aims to document a case of paraplegia associated with IVDH in a
Beagle dog and its therapeutic management using allogenic bone marrow-derived mesenchymal stem
cells (aBM-MSCs).
Case description: The dog was presented with paraplegia that initiated three weeks back. Based on
the findings of computed tomography (CT), the condition was diagnosed as Hansen type I
intervertebral disc herniation at T12-T13 intervertebral space.
Findings/treatment and outcome: Neurological examination was performed to grade the
neurological deficit. The isolation, culture, and characterization of aBM-MSCs were done as per the
standard protocol. The prepared cell suspension of aBM-MSC was percutaneously transplanted to the
spinal cord parenchyma at the site of injury. A total of four doses of 1x106 cells were given at an
interval of 15 days along with methylcobalamin and gabapentin orally. Improvement was evaluated
based on the neurological examination and grading. Significant improvement was noticed after the
first dose of aBM-MSCs. The animal started complete weight bearing on its pelvic limbs after two
doses.
Conclusion: Percutaneous transplantation of aBM-MSC might have played an important role in
reversing the neurological deficits secondary to IVDH in this dog. Further studies are required
preferably in a larger population to confirm the efficacy of aBM-MSC therapy in ameliorating neural
deficits associated with IVDH.
Key words – Mesenchymal Stem Cells; Paraplegia; Intervertebral Disc; Spinal Cord Injuries; Dogs

Abbreviations - aBM-MSC (Allogenic bone marrow-derived mesenchymal stem cell), CT (Computed

tomography), MSC (Mesenchymal stem cell), IVDH (Intervertebral disc herniation), IVDD
(Intervertebral disk disease)

Introduction
Type I intervertebral disk disease (IVDD) is a result of intervertebral disk degeneration that is
severe enough to cause extrusion of the central nucleus pulposus into the vertebral canal. This can
result in compressive injury to the spinal cord and continued compression may cause severe
impairment in neurological function (Gordon-Evans et al., 2019). Degeneration of disc can result in two
types of disc herniation, extrusion and protrusion. The extrusion of intervertebral disc associated with
chondroid degeneration is called as the Hansen Type I lesion. It is commonly observed in
chondrodystrophic dog breeds (Smolders et al., 2013). Intervertebral disc herniations (IVDH) occurs
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Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

most commonly in the thoracolumbar region, causing upper motor neuron (UMN) deficit in the pelvic
limbs (Ruddle et al., 2006). The dogs that are affected by thoracolumbar intervertebral disc disease
may have classical clinical signs that ranges from back pain to paralysis
Diagnosis of IVDH in dogs can be done by using diagnostic modalities like myelography,
computed tomography (CT), and magnetic resonance imaging (MRI). Among these, high-field MRI
is considered to be the ideal modality for diagnosis of dogs with disc disease. Plain CT can be used to
identify the extrusion of mineralized disc material, a condition that is common in chondrodystrophic
dog breeds (Robertson and Thrall, 2011). Surgical management of IVDH involves decompression by
performing hemilaminectomy (Mateo et al., 2019).
The transplantation of BM-MSCs can be considered as a promising, safe, and reliable treatment
strategy for the management of chronic spinal cord injury in canines (Sarmento et al., 2014; Sharun et
al., 2020). Percutaneous transplantation of neurogenically-induced BM-MSCs has been used for the
treatment of paraplegia in dogs secondary to intervertebral disk disease after performing surgical
decompression (Besalti et al., 2015). Dogs with chronic spinal cord injury (SCI) secondary to
thoracolumbar IVDH have been successfully treated by foetal bone marrow stem cells injected
intramedullary immediately following hemilaminectomy (intra-operative transplantation) (Sarmento et
al., 2014). Further, transplantation of adipose-derived mesenchymal stem cells into the injured spinal
cord parenchyma following decompression surgery was associated with better clinical outcome
compared to decompression surgery alone (Kim et al., 2016). Percutaneous transplantation has also
been attempted using autologous enriched olfactory ensheathing cells in dogs with chronic IVDD that
had not received decompressive surgery (Granger et al., 2012).
The aim of this clinical communication is to put on record the diagnosis and treatment of
Hansen type I intervertebral disc herniation (IVDH) without surgical decompression using
percutaneous transplantation of allogenic BM-MSC stem cells in a Beagle dog.

Case Presentation

A four-year-old, male Beagle dog was presented with spontaneously developed paraplegia
three weeks back. The patient was previously treated and was found to be refractory to medical
management using steroids and other supportive therapy. Upon examination, the patient was found to
be active and alert. The animal was dragging its pelvic limbs while walking indicating paraplegia.
Neurological examination revealed the absence of superficial pain perception whereas deep pain was
conserved. Postural reactions like conscious proprioception, wheel barrowing, hopping, tactile and
visual placing were absent in the pelvic limbs. All the spinal reflexes (patellar reflex, flexor reflex, and
perianal reflex) were found to be normal and the animal had voluntary control over urination. The
panniculus (cutaneous trunci) reflex was found to be absent caudal to T12 vertebrae. The dog was
classified under grade 3 (paraplegia with intact deep pain perception) category of neurological signs
as suggested by wheeler and sharp, (1994). Based on the neurological examination, neurolocalization
of the lesion was done to T3-L3 segment of the spinal cord. Further investigations were recommended
to confirm the findings of neurological examination.

3|Page
Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

Diagnostic imaging

Radiographic examination identified reduced disc space between the vertebral body of T12 and
T13 thoracic vertebrae (Figure 1). Computed tomography was performed to localize the site of lesion.
CT scans identified herniated disc material within the vertebral canal of the T12 and T13 thoracic
vertebrae. Mineralized disk material from the T12-T13 intervertebral disk space was found to be
herniating into the vertebral canal, resulting in focal spinal cord compression (Figure 2-3). Hence the
case was diagnosed as paraplegia secondary to Hansen type I intervertebral disc herniation at T12-T13
intervertebral space.

Figure 1 – Lateral radiograph of thoraco-lumbar spine. Reduced intervertebral space between T12 and
T13 thoracic vertebrae (yellow arrows).

4|Page
Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

Figure 2 – Transverse plane CT image showing the presence of extruded disk material in the vertebral
canal of T12 vertebrae (Slice thickness - 2.5 mm, kV - 120, mA - 89). (a) Normal CT image for
comparison. (1) Spinous process (2) Vertebral canal (3) Vertebral body (b) Mineralized disc material
extending into the spinal cord canal. (4) Vertebral lamina (5) Spinal cord (6) Extruded material
pressing on the spinal cord.

Figure 3 – (a) Dorsally reconstructed CT image of the same dog allowing accurate lesion localization
at T12-13 intervertebral space (arrow). (b) Sagittal reconstructed CT image showing dislocated
intervertebral disc material seen as a hyper-attenuating mass in the vertebral canal (arrow).

Treatment

The owner declined the request for surgical decompression due to its invasive nature. Hence,
we suggested stem cell therapy using BM-MSCs as an attempt to reverse the neurological deficit
associated with Hansen type I intervertebral disc herniation. Bone marrow was collected from the
healthy dogs presented to the clinic for elective surgery with the consent of pet owners. The animals
were anesthetized before the collection of bone marrow (iliac crest) as per the protocol described by
Sharun et al. (2020). Protocols used for the isolation and culture of allogenic BM-MSCs were similar
to that performed by Bhat et al. (2018). MSCs at 3rd passage were characterised as per the International
Society for Cellular Therapy criteria and following method of Ansari et al. (2013). Briefly, the growth
characteristics of MSCs were observed for their plastic attachment and fibroblastic growth, trilineage
differentiation potential (osteogenic, adipogenic, and chondrogenic), expression of certain stem cell
surface markers (CD73, CD90, CD 105 and CD34) by reverse transcription polymerase chain reaction
(RT-PCR) and immunocytochemistry. The MSCs were also evaluated for expression of pluripotency
markers Like Nanog, Oct4, SOX2 using RT-PCR assay. Well characterized allogenic canine BM-
MSCs at 3rd passage was ready for use in our laboratory.

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Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

For injecting the stem cells, the dog was anesthetized with xylazine (1 mg/kg body weight)
followed by ketamine (5 mg/kg body weight) intramuscularly. The prepared cell suspension (1ml) of
allogenic BM-MSC containing 1 x 106 cells/ml was percutaneously transplanted to the spinal cord
parenchyma at the site of injury. The animals were positioned in sternal recumbency and a 26-gauge
spinal needle was inserted at T12-T13 interface. It was then directed through the interarcuate space till
it penetrated the spinal cord parenchyma which was evident from the sudden jerk produced by the
lightly anaesthetized animal. After the tip has entered parenchyma, half of the stem cells were injected
slowly into the spinal cord. The remaining half was injected while withdrawing the needle from the
spinal cord parenchyma to deposit the cells at multiple layers. A total of four doses where given at an
interval of 15 days along with supportive therapy that included methylcobalamin (500mcg/dog q12
hour) and gabapentin (10 mg/kg body weight q12 hour) orally throughout the study period.
Improvement was evaluated based on the neurological examination and grading. Recovery was scored
using the scale (on a scale of 0 to 14) suggested by Olby et al., (2001).

Figure 4 – Graph plotting the progressive improvement of neurological function of the dog. Significant
improvement in Olby score was observed immediately after the first BM-MSC injection. Complete
weight bearing was observed after two doses.

Result

Significant improvement was noticed after the first dose of aBM-MSCs. The animal started
complete weight bearing on its hind limbs after two doses. Improvement was also observed in the
6|Page
Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

neurological score which indicated improvement in sensory function. Progressive improvement in the
neurological function was evident from the increased recovery score (Figure 4-5). Excellent
improvement was observed in pain perception (nociception) that led to return of superficial pain
perception. Gradual improvement in postural reactions like conscious proprioception, visual/tactile
placing, and wheel barrowing were also recorded.

Figure 5 - (a) Day 0 – Paraplegia with dragging of hind limbs. (b) Day 30 – Initiated weight bearing
on the hind limbs. (c) Day 45 – Weight bearing on hind limbs that has normal strength, but had ataxia.
(d) Day 60 – Almost normal gait with mild ataxia during walking. (e) Day 90 – Complete recovery
with return of normal behaviour (scratching of ear with hind limbs). (f) Day 90 – Dog started moving
up and down the stairs with ease.

Discussion

CT images of type I disk extrusion are characterized by the presence of hyper-attenuating disk
material in the epidural space. The density of the extruded disk material is directly dependent upon the
degree of mineralization. The disk material can either migrate horizontally along the vertebral canal
floor and circumferentially around the spinal cord or can migrate dorso-laterally into the intervertebral
foramina. Extruded disc can displace and compress the spinal cord depending on the volume and
distribution of disk material (Wisner and Zwingenberger, 2015). The calcified disc, before extrusion can
be visualized in the disc space. Whereas the extruded calcified disc material will be visible at the
intervertebral foramen above the disc. Once it is extruded, the amount of calcified disc material
remaining in the disc space is less and hence cannot be detected using radiography (Stigen et al., 2019).
In the present case, radiographs couldn’t identify the presence of any calcified disc material in the T12-
T13 interface but significant reduction in the disc space was observed. Narrowing of disc space cannot

7|Page
Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

be considered as a sign of disc herniation since the disc can herniate laterally, dorsally as well as
ventrally. Extruded disc material may not be visible radiographically as radiography had less
sensitivity compared to CT in detecting calcified disc material in the vertebral canal. But radiograph
may exhibit higher sensitivity in detecting remaining calcified material in the disc space (Stigen et al.,
2019). It is also very difficult to identify calcified disc in thoracic vertebrae since the opacification of
intervertebral foramina occurs naturally due to the superimposition of ribs.

Treatment options for IVDH involves the use of corticosteroids, acupuncture, decompressive
surgical techniques, cage rest, or can even be a combination of the aforementioned options. Surgical
decompression is the established treatment for non-ambulatory dogs which involves laminectomy
followed by the removal of disc material from the spinal canal (Ruddle et al., 2006). Surgical
management of disc extrusions has higher success rate in dogs with intact pain perception when
compared to dogs that lacked conscious pain sensation. But the neurological recovery following
surgical management in IVDH can be time consuming, expensive, and frustrating to the owners (Olby
et al., 2003; Ruddle et al., 2006). Percutaneous cellular therapy using autologous enriched olfactory
ensheathing cells have been reported to have therapeutic potential in chronic spinal cord injury
(Granger et al., 2012). Here we have chosen allogenic BM-MSCs for the percutaneous implantation
without performing any decompressive surgery.

Compared to dogs that had traumatic SCI, the prognosis for motor function recovery is
significantly higher in dogs that had SCI secondary to IVDH. This can be attributed to the fact that
vertebral fracture or luxation are associated with spinal cord lacerations in addition to
contusive/compressive injuries (Olby et al., 2003). In the present case Hansen type I intervertebral disc
herniation at T12-T13 was associated with grade 3 injury characterized by paraplegia with intact deep
pain perception. The mild nature of the injury might have contributed to the complete neurological
recovery without the need of any surgical decompression. Stem cell therapy might have played an
important role in the sudden recovery in neurological function. The mesenchymal stem cells (MSCs)
isolated from different sources not only have the ability to differentiate into multiple cell lineages but
also has several trophic, anti-inflammatory, and regenerative effects (Figueroa et al., 2012). The
transplantation of MSCs is associated with improved clinical signs due to their ability to modify
inflammatory environment that promotes the survival of endogenous nerve cells. MSCs also modulates
various regulatory signal molecules thereby reducing glial scar formation (Kim et al., 2016). They are
also reported to restore the myelination of injured axons. The MSCs possess immunosuppressive
properties and also secrete certain growth/trophic factors that promotes regenerative processes
(Meirelles et al., 2009). The present case that was refractory to the routine treatment responded to stem
cell therapy very quickly proving the efficacy of stem cell therapy in cases of IVDH, with intact deep
pain sensation.

The prognosis of cases affected with IVDH are reported to be greatly dependant on the presence
or absence of deep pain perception (Olby et al., 2003; Ruddle et al., 2006). The absence of deep pain
perception following SCI is indicated as the functional spinal cord transection (Olby et al., 2003).
Studies have shown that those patients that lacked deep pain perception were 1.7 times less likely to
8|Page
Citation: Sharun K, Kumar R, Chandra V, Saxena AC, Pawde AM, Kinjavdekar P, Dhama K, Amarpal, Sharma
GT. Percutaneous transplantation of allogenic bone marrow-derived mesenchymal stem cells for the
management of paraplegia secondary to Hansen type I intervertebral disc herniation in a Beagle dog. Iran J Vet
Res. 2020, (In Press). DOI: 10.22099/IJVR.2021.38613.5620

become ambulatory following the treatment (Ruddle et al., 2006). The reason for the good prognosis in
this case can be attributed to the conservation of deep pain perception even though superficial pain
perception were lacking.

Conclusion

In conclusion, percutaneous transplantation of aBM-MSCs might have helped the dog to regain
its neurological function completely without performing surgical decompression. Percutaneous
delivery of stem cells was preferred to avoid the need of any surgical intervention. Hence this non-
invasive technique may be further evaluated for its efficacy in managing paraplegia secondary to
Hansen type I IVDH. Further evaluation is required in a larger cohort of dogs to determine the efficacy
and success rates associated with this procedure.

Funding

Authors received no funding/grant support for this study.

Ethics approval

Written consent was obtained from the owner before performing the therapy. All procedures performed
are in accordance with the ethical standards of the institution at which the studies were conducted.

Conflict of interest

None of the authors has a financial or personal relationship with other people or organizations that
could inappropriately influence or bias the content of the paper.

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